Methods of delivering anionic agents in vivo using non-viral nanoparticle-based delivery systems

ABSTRACT

The present invention concerns a polymeric material for the production of a non-viral nanoparticle. The polymeric material comprises (i) a hydrophilic linear polymer having a first end and a second end, (iii) a cross-linkable cationic polymer covalently bonded to the first end of the hydrophilic linear polymer, and (iii) at least one targeting/penetrating peptide covalently associated to the second end of the hydrophilic linear polymer. Also disclosed herein are nanoparticles produced with these polymeric material, processes for making the polymeric material and the nanoparticles as well as use of the nanoparticles.

CROSS-REFERENCE TO RELATED APPLICATIONS AND DOCUMENTS

This application is a continuation of U.S. utility patent applicationSer. No. 16/218,347, filed Dec. 12, 2018, now U.S. Pat. No. 10,799,601,which is a continuation of U.S. utility patent application Ser. No.15/335,408, filed Oct. 26, 2016, now abandoned, which is a continuationof and claims priority under 35 U.S.C. § 120 from application Ser. No.13/916,941 filed Jun. 13, 2013, now abandoned, which claims priorityfrom U.S. provisional patent application 61/660,139 filed on Jun. 15,2012. The contents of these priority applications are incorporatedherein in their entirety. A sequence listing in a computer readableformat is enclosed and incorporated herein in its entirety.

TECHNOLOGICAL FIELD

The present invention relates to the development of nanoparticles fordelivery of anionic agents such as, for example, nucleic acids, drugsand other molecules. The non-viral nanoparticle design involves thechemical modification of a cationic cross-linkable polymer (such aschitosan) with a hydrophilic linear polymer (such as polyethyleneglycol) and a targeting/penetrating peptide. The nanoparticle system canbe used for in vitro applications as well as in vivo applications.

BACKGROUND

Delivering agents specifically to a tissue, an organ or a cell type invivo is a complex task. However, such specific delivery may be crucialfor providing therapeutic benefits and/or limiting undesirable sideeffects. In addition, some agents may also benefit from being moreefficiently translocated across a biological membrane and/or a cellularmembrane (such as, for example, the cytoplasmic membrane or the nuclearmembrane).

One application where specific drug-delivery and/or upregulated cellularuptake is important is neurodegenerative diseases. Neurodegenerativediseases are characterized by progressive, age-related loss of specificsubsets of neural cells, which lead to diverse clinical phenotypesdepending on the underlying anatomical involvement. The etiology ofneurodegenerative diseases is most often multifactorial, likely a resultof gene—environmental interaction, which may lead to diseases likeParkinson's, Alzheimer's, Huntington's, Amyotrophic lateral sclerosis(ALS) and Spino Cerebellar Ataxia (SCA). These diseases tend to progressslowly over the time and generally target older population. Currently,there is no treatment available for many of the neurodegenerativediseases that may halt the progression of the disease. The currentclinical strategy to deliver drug/gene to the central nervous systeminvolves surgical interventions, which can later pose surgicalcomplications like fluid retention in the ventricles etc., which canhave fatal side-effects. It has been estimated that up to 98% of thenewly developed small molecules will not/cannot cross the blood-brainbarrier (BBB). Also, it is challenging to achieve sufficientdistribution and diffusion of the therapeutic drug.

Another application where specific drug-delivery and/or upregulatedcellular uptake is important is cancer. Cancer is characterized byuncontrolled growth of a group of cells that infests adjacent tissuesand often metastasizes to other organs via lymphatic system or bloodstream. Cancer is primarily caused by environmental factors (90-95%) andfew with genetic (5-10%). The uncontrolled growth of group of cells inthe case of cancer is usually triggered by malfunctioning of the genesthat manipulate cell's growth and differentiation. Typically thealteration in cell growth promoting, oncogenes and cell divisioninhibiting, tumor suppressive genes lead to the formation of cancercells. The genetic causes of cancer are usually due to gain or loss ofan entire chromosome due to errors in mitosis or changes in nucleotideleading to mutations in the genomic DNA. Depending on the stage of thecancer the treatment options available include surgical removal,chemotherapy with anticancer drugs, such as 5-fluorouracil, oxaliplatinand leucovorin, radiation therapy, immunotherapy and hormone blocktherapy with drugs like cetuximab and panitumumab. However, it has beenshown that cancers with genetic origin are not benefited with thesechemotherapies. Moreover, the toxicity and side effects have severelylimited the safety and effectiveness of these methods.

RNA interference has been demonstrated as an effective/novel therapeuticmodality in vivo for the reduction of pathological molecules in neurons,leading to significant efficacy in animal models of Alzheimer's disease,ALS, anxiety, depression, encephalitis, Huntington's disease,neuropathic pain and spinocerebellar ataxia. In addition, siRNAstargeted against proliferation-associated signal transduction pathways,which can halt the tumor progression in animal models, is also emergingas an appealing approach for cancer therapy. SiRNA mediated gene therapyis further being explored by using it in combination with antineoplasticagents as a combinatorial therapy towards cancer treatment.

The therapeutic delivery across BBB and physiological barriers of smallmolecules like siRNA/nucleic acids/proteins and drugs is challenging orrather ineffective owing to their instability in physiologicalconditions, improper cellular distribution, low bioactivity, high dosagerequirement, and the necessity for continuous long-term infusions.Moreover, off-target effects of siRNA have shown to induce and activateimmune responses through Toll-like receptor (TLR)-dependent and TLRindependent mechanisms, which can cause toxicological effects due to theactivation of interferons (IFNs) and inflammatory cytokines. Though,this strategy can be used as a therapeutic option for cancer therapy, itcan also lead to unwanted toxicological effects on non-cancerous cells.The application of a novel therapeutic modality highly depends on thedevelopment of an efficient and clinically feasible means of safeadministration. Recent advancements in siRNA mediated gene therapy isfocusing on the incorporation of specific nucleotide chemistries in thesiRNA sequence, such as 2′-O-methyl-modified nucleotides, in order toimprove its pharmacologic and nuclease resistant properties and lowimmuno-stimulatory effect. However, to streamline the systemic deliveryof siRNAs in vivo to a specific target tissue, a site-directed deliverystrategy is essential.

Various commercially available delivery/transfection reagents do fulfillthe need of siRNA delivery in substantially lower doses than compared tosiRNA delivered alone, but they lack to emphasize on issues such astarget specificity, cytotoxicity, immunological responses, stablesystemic delivery and off-target effects of these reagents. Moreover,the efficacy of most of these commercially available transfectionreagents is limited to in vitro use. In addition to transfectionreagents, viral gene therapy vectors have shown to be most effective butconcerns regarding their safety, immunogenicity has limited their use.Moreover, for in vivo applications, delivery via systemic route targetsmultiple sites, which may not be an ideal deal for many biomedicalapplications. Thus, development of a delivery vehicle that can addressthese issues and identify cell-specific receptors, with an ability todistinguish between diseased and normal cells and efficiently delivers asufficient dose of siRNA to the intracellular compartment of the celltype of interest within the target tissue will be an effective approachto overcome the limitations of currently used therapeutics in in vivomodels.

Cationic biodegradable polymers, such as polyethyleneimine (PEI), arecommonly used for siRNA transfection, providing a positively chargedvehicle to carry the negatively charged siRNA into the target cells.Most of the nanoparticles that are widely being accepted as analternative approach to gene delivery are developed from cyclodextrinpolycations, poly-L Lysine, polyamidoamines, chitosan, quantum dots andliposomes to deliver siRNA for on-target gene silencing. However,transfection frequency, cytotoxicity, serum stability, and activetargeting are some of the major concerns associated with non-viral modeof delivery. To overcome the aforementioned effects of nanoparticles,they are surface coated with PEG to eliminate phagocyte capture andnonspecific immune stimulation, which provides better stability andextended blood circulation with low toxicity. Conjugating siRNA withcholesterol and transfection enhancers, such as penetratin1, providessubstantial advantage for in vivo delivery and bioactivity of siRNA.

Lipid nanoparticles have been used for drug delivery targeting brain.However, such formulations lack active targeting to the tissue ofinterest. It has been proposed to use monoclonal antibody as a targetingmoiety on a non-viral liposome nanoparticles targeting brain. However,the high molecular weight of antibodies makes the overall size of thenanoparticles big in size, which can therefore be a hindrance to achieveoptimal biodistribution at the targeted site. A recent addition to theart is the use of PEI and nucleic acid complex for intraventricularstereotactic screening of stem cells of the brain as a treatment forneurodegenerative diseases and demyelinating diseases. However, PEI ashas been known to be a non-biodegradable and toxic could be replaced bybiodegradable nanocomplexes with cell-targeting ligands.

In summary, it would be highly desirable to be provided with a deliverysystem for specifically delivering agents to a tissue (or a group oftissues), an organ (or a group of organs) or a cell type (or a group ofcell types). The cellular specificity of the delivery system could beused to lower the dose of the agent as well as limit, if any, theundesirable effects associated with the agent. The delivery system ispreferably not based on a viral system and is not capable of reproducingonce it enters a cell. It would also be desirable that the deliverysystem be used in vitro for research purposes, ex vivo for cellulartherapy applications as well as in vivo for therapy or diagnostics. Insome embodiments, it would be desirable to be provided with a deliverysystem that is also capable of more efficiently translocating across abiological membrane and/or a cellular membrane (such as, for example,the cytoplasmic membrane or the nuclear membrane).

SUMMARY

According to a first aspect, the present invention provides a polymericmaterial of a non-viral nanoparticle. The polymeric material comprises(i) a linear polyethylene glycol polymer having a first end and a secondend, (ii) a cross-linkable cationic chitosan polymer covalentlyassociated to the first end of the linear polyethylene glycol polymer,and (iii) at least one targeting/penetrating peptide covalentlyassociated to the second end of the linear polyethylene glycol polymer.In one embodiment, the linear polyethylene glycol polymer has an averagemolecular weight between about 1000 to about 5 000 Da. In anotherembodiment, the linear polyethylene glycol polymer has an averagemolecular weight of about 2 000 Da. In still another embodiment, thecross-linkable cationic chitosan polymer has an average molecular weightof about 10 to about 200 000 Da. In still a further embodiment, theaverage molecular weight of the cross-linkable cationic chitosan polymeris about 1 00 000 Da. In one embodiment, the cross-linkable cationicchitosan polymer has a degree of deacetylation of about 75 to about 85%.In another embodiment, the degree of deacetylation of the cross-linkablecationic chitosan polymer is about 80%. In yet another embodiment, theat least one targeting/penetrating peptide can bind to a cell surface orcell-associated receptor, such as, for example, a targeting peptidehaving an amino acid sequence comprisingNH₂-YQPPSTNKNTKSQRRKGSTFEEHK-NH₂ (MGF) (SEQ ID NO: 1), a targetingpeptide having an amino acid sequence comprising NH₂-VHLGYAT-NH₂ (CP15)(SEQ ID NO: 2) and/or or a targeting peptide having an amino acidsequence comprising NH₂-VPWMEPAYQRFL-NH₂ (P160) (SEQ ID NO: 3). Inanother embodiment, the at least one targeting/penetrating peptide canfacilitate the translocation of the non-viral particle across abiological or a cellular membrane, such as, for example, a penetratingpeptide having an amino acid sequence comprising NH₂-RKKRRQRRR-NH₂ (TAT)(SEQ ID NO: 4).

In a second aspect, the present invention provides a process for makingthe polymeric material described herein. Broadly, the process comprises(i) reacting the first end of a linear polyethylene glycol polymerderivative with the cross-linkable cationic chitosan polymer to form acovalent bond between the cross-linkable cationic chitosan polymer andthe linear polyethylene glycol polymer, and (ii) reacting the second endof the linear polyethylene glycol polymer derivative with the at leastone targeting/penetrating peptide to form a covalent bond between the atleast one targeting/penetrating peptide and the linear polyethyleneglycol polymer. In an embodiment, the process further comprises, in step(i) prior to reacting the first end, protecting at least one amine groupof the cross-linkable cationic chitosan polymer. In still anotherembodiment, the process further comprises, after step (ii), deprotectingthe at least one amine group of the cross-linkable cationic chitosanpolymer. In an embodiment, the linear polyethylene glycol polymerderivative is a linear monomethyl ether polyethylene glycol (mPEG-OH).In another embodiment, the process further comprises, in step (i) priorto reacting the first end, activating the first end of the linearpolyethylene glycol monomethyl ether by reacting the polyethylene glycolmonomethyl ether with succinic anhydride to form mPEG-COOH and reactingthe mPEG-COOH with thionyl chloride to form a mPEG-COCl. In stillanother embodiment, the process further comprises, in step (i), reactingthe mPEG-COCl with the cross-linkable cationic chitosan polymer to forma PEGylated chitosan. In yet another embodiment, the process furthercomprises, in step (ii) prior to reacting the second end, activating thesecond end of the polyethylene glycol monomethyl ether by reacting themethyl ether group of the polyethylene glycol with aluminium chloride inethanethiol to convert the methyl ether group to a hydroxyl group andreacting the hydroxyl group of the polyethylene glycol with succinicanhydride to form an activated PEG-COOH. In an embodiment, the processfurther comprises, in step (ii), reacting the activated PEG-COOH withthe at least one targeting/penetrating peptide to form the polymericmaterial.

According to a third aspect, the present invention provides a non-viralnanoparticle. The non-viral nanoparticles comprises (i) a plurality ofpolymeric materials as described herein or produced by the processdescribed herein, wherein the cationic polymer is cross-linked so as toform an internal core; and (ii) an anionic agent entrapped in theinternal core. In an embodiment, the anionic agent is a nucleic acidmolecule, such as, for example, a short interfering RNA (siRNA). In anembodiment, the non-viral nanoparticles have a diameter of less than 5nm. In another embodiment, the non-viral nanoparticles have a diameterbetween 5 and 10 nm. In a further embodiment, the non-viralnanoparticles have a diameter between 100 and 200 nm. In still anotherembodiment, the non-viral nanoparticle have a diameter between about 5nm to about 100 nm or between about 50 nm to about 300 nm. In yetanother embodiment, the plurality of polymeric materials comprises afirst polymeric material having a first targeting/penetrating peptidethat can bind to a cell surface or cell-associated receptor and a secondpolymeric material having a second targeting/penetrating peptide thatcan facilitate the translocation of the non-viral particle across acellular membrane. In such embodiment, the first targeting/penetratingpeptide can have an amino acid sequence comprisingNH₂-YQPPSTNKNTKSQRRKGSTFEEHK-NH₂ (MGF) (SEQ ID NO: 2) and/or the secondtargeting/penetrating peptide can have an amino acid sequence comprisingNH₂-RKKRRQRRR-NH₂ (TAT) (SEQ ID NO: 4).

According to a fourth aspect, the present invention provides a processfor making a non-viral nanoparticle. Broadly, the process comprises (i)admixing an anionic agent with a cross-linker to form a first solution;and (ii) adding the first solution to a second solution comprising aplurality of polymeric materials described herein or produced by theprocess described herein so as to form the non-viral nanoparticle. In anembodiment, the cross-linker is sodium tripolyphosphate (TPP). Inanother embodiment, the anionic agent is a nucleic acid molecule, suchas, for example, a short interfering (RNA). In an embodiment, thenon-viral nanoparticle has a diameter between about 5 nm to about 100 nmor between about 50 nm to about 300 nm. In an embodiment, the non-viralnanoparticles have a diameter of less than 5 nm. In another embodiment,the non-viral nanoparticles have a diameter between 5 and 10 nm. In afurther embodiment, the non-viral nanoparticles have a diameter between100 and 200 nm. In still another embodiment, the plurality of polymericmaterials comprises a first polymeric material having a firsttargeting/penetrating peptide that can bind to a cell surface orcell-associated receptor and a second polymeric material having a secondtargeting/penetrating peptide that can facilitate the translocation ofthe non-viral particle across a cellular membrane. In such embodiment,the first targeting/penetrating peptide can have an amino acid sequencecomprising NH₂-YQPPSTNKNTKSQRRKGSTFEEHK-NH₂ (MGF) (SEQ ID NO: 1) and/orthe second targeting/penetrating peptide can have an amino acid sequencecomprising NH₂-RKKRRQRRR-NH₂ (TAT) (SEQ ID NO: 4).

According to a fifth aspect, the present invention provides a method ofdelivering an anionic agent to a cell. Broadly the method comprisescontacting the non-viral nanoparticle described herein or produced bythe process described herein with the cell under conditions sufficientfor allowing the anionic agent to enter the cell. In an embodiment, thecell is in vitro. In another embodiment, the cell is to be introducedinto an individual in need thereof. In still another embodiment, whereinthe cell is in an individual in need thereof. In an embodiment, thenon-viral nanoparticle is formulated for intranasal administration priorto being administered to the individual, such as, for example, as anintranasal drop. In another embodiment, the method is for the deliveryof the agent to the brain. In another embodiment, the method is for theprevention, treatment and/or alleviation of symptoms associated to aneurodegenerative disease or a brain cancer. In some embodiments, theneurodegenerative disease is selected from the group consisting orspinocerebellar ataxia, Huntington's disease, Parkinson's disease,Alzheimer's disease, dementia and amyotrophic lateral sclerosis. Inother embodiment, the non-viral nanoparticle is formulated forintravenous administration prior to being administered to theindividual. In another embodiment, the method is for the prevention,treatment and/or alleviations of symptoms associated with aproliferative disease, such as, for example cancer (e.g. colon cancer,breast cancer or brain cancer).

Throughout this application, various terms are used and some of them aremore precisely defined herein.

Anionic agent. The anionic agent bears a net negative charge (at aphysiological pH) and can be formulated inside the internal core formedby the cross-linked cationic polymer. In one embodiment, it is atherapeutic agent which can mediate a therapeutic/biological action on acell or in an organism. In another embodiment, it is a diagnostic agentwhich can specifically be located in a cell or in a tissue fordiagnostic purposes (e.g. imaging for example). The anionic agent can bea nucleic acid molecule, such as a RNA or a DNA. In some embodiments,the anionic agent can be, for example an small interfering RNA (siRNA),short hairpin (shRNA), ribozymes, micro RNA (miRNA), triplexoligonucleotides, antisense oligonucleotides, plasmid DNA (pDNA) as wellas combinations thereof. In another embodiment, the anionic agent is asmall chemical entity, such as a drug (e.g. an anti-cancer drug).

Cross-linkable cationic polymer. This polymer bears a net positive andis capable of being cross-linked. Its cross-linkability may be inherentto the polymer (e.g. the polymer may itself contain cross-linkablegroups). However, the polymer can also be modified to augment itscross-linkability. The cross-linkable moieties of the polymer arepreferably amino groups. This polymer is preferably biocompatible. Thispolymer, in some embodiments, can be considered hydrophobic or slightlysoluble in water. Once integrated in the polymeric material or thenanoparticle, the polymer preferably is not cytotoxic. In an embodiment,the cationic polymer is chitosan.

Chitosan. Chitosan is a polysaccharide obtained by N-deacetylation ofchitin. In industrial scale procedures, chitosan is obtained from chitinby alkali treatment of crustacean shells. Chitosan is also present innature in the cell walls of some fungi and algae and in insects.Chitosan is mainly composed of β-1,4-linked D-glucosamine units with avariable content of N-acetyl-D-glucosamine units. The percentage ofN-acetyl-D-glucosamine units is defined as the degree of N-acetylationof chitosan (“DA”), while the percentage of D-glucosamine units is alsocalled the degree of deacetylation (“DDA”) of chitosan. Most commercialpreparations of chitosan are characterized by dda values between 70 and99%. Native chitosan molecules, as isolated from natural organisms orobtained after alkaline N-deacetylation of chitin, are of high molecularweight (in the range of millions of daltons) with degrees ofpolymerization reaching several thousand units. While variousapplications were described for such high molecular weight chitosans(HMWC), for most applications a narrower, optimal range of molecularweight is under consideration. Chitosan polymers with shorter thannative chains are often divided into low molecular weight chitosans(LMWC), with a range of molecular weight roughly between 5 kDa and 100kDa and chitooligosaccharides (or chitosan oligosaccharides; CHOS) witha lower limit of 0.4 kDa (glucosamine dimer) while the higher limit isless defined (5 to 10 kDa). The CHOS are fully soluble in water and areessentially prepared as undefined mixtures of oligomeric molecules ofvarious molecular weights and degrees of N-acetylation. Chitosan is acationic polymer due to the presence of amine groups at the C6 positionof its pyranose ring and, in its native unmodified state, is onlysoluble in mild acidic conditions.

Crosslinker. The crosslinker is a compound that promotes or regulatesthe cross-linking between different groups of the cationic polymerchains, linking them together to create an internal core. In anembodiment, the crosslinker is sodium tripolyphosphate (TPP).

Hydrophilic linear polymer. This polymer is linear in nature (it lacksany branching and only possesses two opposite terminal ends). Thehydrophilic linear polymer has at least two distinct and different end,each covalently associated with either the cationic polymer or thetargeting/penetrating peptide. It also acts as a linker to camouflagethe cross-linked cationic polymer internal core. It also serves topresent the targeting/penetrating peptide to the cell. This polymer ispreferably hydrophilic to facilitate cellular membrane translocation ofthe non-viral nanoparticle. This polymer can be biophobic (does notallow on its own, the association to a cell). This polymer is preferablybiologically inert.

Internal core. This section of the nanoparticle is formed once thecationic polymer has been cross-linked. The internal core is camouflagedunder a layer of hydrophilic linear polymer and contains the anionicagent.

Non-viral nanoparticles. As used in the context of this invention, theterm “non-viral nanoparticles” refer to particles in the nanometerrange. These particles are considered to be “non-viral” because, eventhough in some embodiments they are can contain nucleic acid molecules,the particles are not capable of replicating once they have entered ahost cell. These nanoparticles are generally of a spherical shape. Insome embodiments, the average diameter of the non-viral nanoparticle canbe between about 5 to about 100 nm or to about 200 nm. In otherembodiments, the average diameter of the non-viral diameter can betweenabout 50 to about 300 nm. In an embodiment, the non-viral nanoparticleshave a diameter of less than 5 nm. In another embodiment, the non-viralnanoparticles have a diameter between 5 and 10 nm. In a furtherembodiment, the non-viral nanoparticles have a diameter between 100 and200 nm.

Pharmaceutical composition. As used herein, “pharmaceutical composition”means therapeutically effective amounts (dose) of the non-viralnanoparticles together with pharmaceutically acceptable diluents,preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.Such compositions are liquids or lyophilized or otherwise driedformulations and include diluents of various buffer content (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength, additives such asalbumin or gelatin to prevent absorption to surfaces, and detergents(e.g. Tween 20™, Tween 80™, Pluronic F68™, bile acid salts). Thepharmaceutical composition can comprise pharmaceutically acceptablesolubilizing agents (e.g. glycerol, polyethylene glycol), anti-oxidants(e.g. ascorbic acid, sodium metabisulfite), preservatives (e.g.thimerosal, benzyl alcohol, parabens), bulking substances or tonicitymodifiers (e.g. lactose, mannitol), covalent attachment of polymers suchas polyethylene glycol to the protein, complexation with metal ions, orincorporation of the material into or onto particulate preparations ofpolymeric compounds such as polylactic acid, polyglycolic acid,hydrogels, etc., or onto liposomes, microemulsions, micelles,unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroplasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance.

Polyethylene Glycol. Polyethylene glycol has the formulaH(OCH₂CH₂)_(n)OH, wherein n is greater than or equal to 4, with amolecular weight of up to about 20 000 Daltons. Various derivatives ofpolyethylene glycol may substitute for the H or OH end groups, forming,for example, polyethylene glycol ethers (e.g. PEG-O-R; PEG-O-CHs;CHs-PEG-OH); 2,4-dinitrophenyl ethers of PEG), polyethylene glycolesters (e.g. PEG-O₂C(CH₂)₁₄CH₃; PEG-O₂CCH₂CH₂CO₂-atropine), polyethyleneglycol amides (e.g. PEG-O₂C(CH₂)₇CONHR;mPEG-O₂CCH₂CH₂CONH(CH₃)CHCH₂C₆H₅; PEG-O₂CCH₂CH₂CONHCH₂CH₂-NAD+),polyethylene glycol amines (e.g. PEG-NH₂; PEG-NH(CH₂)₆NH₂;PEG-OCH₂CH₂NH₂; mPEG-NH₂), polyethylene glycol acids (e.g.PEG-O₂C(CH₂)₂CO₂H; PEG-O-CH₂CO₂H; PEG-O₂C—(CH₂)₇—CO₂H), polyethyleneglycol aldehydes (e.g. PEG-O—CH₂—CHO), and electrophilic derivatives(e.g. PEG-Br; PEG-OSO₂CH₃; PEG-O). Various phenyl moieties can also besubstituted for the H or OH of PEG, such as the 2,4-dinitrophenyl etherof PEG mentioned above. The particular polyethylene glycol derivativeslisted above are exemplary only, and the invention is not intended to belimited to those particular examples.

Prevention, treatment and alleviation of symptoms. These expressionsrefer to the ability of a method or an agent to limit the development,progression and/or symptomology of a specific disorder or pathology. Theprevention, treatment and/or alleviations of symptoms associated with aneurodegenerative disease can encompass the limitation ofneurodegeneration (e.g. by reducing the apoptosis of neuronal cells).The prevention, treatment and/or alleviation of symptoms ofproliferative disease can encompass the reduction of proliferation ofthe cells (e.g. by reducing the total number of cells in anhyperproliferative state and/or by reducing the pace of proliferation ofcells). Symptoms associated with proliferation-associated disorderinclude, but are not limited to: local symptoms which are associatedwith the site of the primary cancer (such as lumps or swelling (tumor),hemorrhage, ulceration and pain), metastatic symptoms which areassociated to the spread of cancer to other locations in the body (suchas enlarged lymph nodes, hepatomegaly, splenomegaly, pain, fracture ofaffected bones, and neurological symptoms), and systemic symptoms (suchas weight loss, fatigue, excessive sweating, anemia and paraneoplasticphenomena).

Proliferation-associated disorders. These disorders form a class ofdiseases where cells proliferate more rapidly, and usually not in anordered fashion. The proliferation of cells cause a hyper-proliferativestate that may lead to biological dysfunctions, such as the formation oftumors (malignant or benign). One of the proliferation-associateddisorder is cancer. Also known medically as a malignant neoplasm, canceris a term for a large group of different diseases, all involvingunregulated cell growth. In cancer, cells divide and growuncontrollably, forming malignant tumors, and invade nearby parts of thebody. The cancer may also spread to more distant parts of the bodythrough the lymphatic system or bloodstream. In an embodiment, thecancer is a carcinoma (such as a colon carcinoma or a breast carcinoma).In another embodiment, the cancer is a glioma (such as a brain glioma).In another embodiment, the cancer is a colon cancer, a breast cancerand/or a brain cancer.

Targeting/penetrating peptide. This peptide is covalently associatedwith the hydrophilic linear polymer and serves to provide specificityfor the delivery of the anionic agent. In some embodiments, thetargeting/penetrating peptide is hydrophilic in nature. For example, atargeting/penetrating peptide can have both polar and apolar ends or itcan have an alternating pattern of hydrophilic and hydrophobic aminoacids. The targeting/penetrating peptide is preferably at least 3 aminoacid-long and less than 40 amino-acids long. In one embodiment, thetargeting/penetrating peptide preferably and specifically binds to acellular structure (such as a cellular receptor having at least oneportion either embedded in the cellular membrane or protruding from thecellular membrane). When the anionic agent is to be administered to aspecific type of cells, the targeting/penetrating peptide specificallybinds to this specific type of cells. Alternatively or complementarily,the peptide may also possess a biological characteristic that enables itto penetrate a certain cellular structure. Specifictargeting/penetrating peptides are known in the art and can be, forexample, capable of penetrating a cellular membrane (such as thecytoplasmic membrane or the nuclear membrane). Sometargeting/penetrating peptide can be specific or derived from a proteintransduction domain. Other targeting/penetrating peptide can be specificor derived from a growth factor or a hormone. An exemplarytargeting/penetrating peptide can be a blood-brain-barrier(BBB)-permeant, amyloid-targeting/penetrating peptide such asKKLVFFAEKGC (SEQ ID NO:16) (as presented in US Patent Serial Number7,803,351). Other exemplary targeting/penetrating peptides include, butare not limited to TAT, MGF, CP15 and P160 as well as combinationsderived therefrom (for example a TAT/MGF combination).

Therapeutically effective amount. A “therapeutically effective amount”as used herein refers to that amount which provides a therapeutic effectfor a given condition and administration regimen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a possible synthetic scheme for preparing peptide-taggedPEGylated chitosan polymer.

FIGS. 2A-2B shows FTIR spectra of commercially available chitosan (CS)(plot a) and 2-N-phthaloylated chitosan: v_(max)/cm⁻¹ 3200-3400 (OH),1774, 1710 (carbonyl anhydride), 1150-1000 (pyranose), and 720 (arom)(plot b).

FIGS. 3A-3B shows FTIR spectra of polyethylene glycol monomethyl ether(mPEG-OH) (plot a) and carboxylated mPEG (mPEG-COOH): v_(max)/cm⁻¹ 1732(C═O), 2878 (C—H stretching), 1100 (C—O stretching) (plot b).

FIGS. 4A-4C shows FTIR spectra of 2-N-phthaloyl chitosan-O-PEG:v_(max)/cm⁻¹ 2873 (C—H stretching), 1068 (C—O stretching) of PEG, 1774,1710 (carbonyl anhydride) and 720 (arom) of phthalimido group onchitosan (plot a); TAT tagged PEGylated phthaloyl chitosan(2-N-phthaloyl chitosan-O-PEG-CONH-TAT): v_(max)/cm⁻¹2919 (C—Hstretching), 1067 (C—O stretching) of PEG, 1774, 1710 (carbonylanhydride) and 720 (arom) of phthalimido group on chitosan, 1659(amides) in TAT peptide (plot b); deprotected TAT tagged PEGylatedchitosan (chitosan-O-PEG-CONH-TAT): v_(max)/cm⁻¹2918 (C—H stretching),1061 (C—O stretching) of PEG, 1644 amides of TAT peptide, and 1543(amides) in chitosan (plot c).

FIGS. 5A-5E shows ¹H NMR spectra of TAT peptide (plot a); deprotectedTAT tagged PEGylated chitosan (CS-O-PEG-CONH-TAT) (plot b); TAT taggedPEGylated phthaloyl chitosan (PHCS-O-PEG-CONH-TAT) (plot c); PEGylatedphthaloyl chitosan (PHCS-O-PEG) (plot d); and phthaloylated chitosan(PHCS) (plot e).

FIGS. 6A-6F show transmission electron microscopy (TEM) image ofPEGylated chitosan polymer, magnification: 53 800×, scale bar: 500 nm(FIG. 6A); TAT/MGF tagged PEGylated chitosan polymer, magnification: 53800×, scale bar: 500 nm (FIG. 6B); acid treated (pH 5.5) TAT/MGF taggedPEGylated chitosan polymer, magnification: 70 700×, scale bar: 500 nm(FIG. 6C); empty TAT/MGF tagged PEGylated chitosan nanoparticles,magnification: 162 000×, scale bar: 100 nm (FIG. 6D); unmodifiedchitosan-siRNA nanoparticles, magnification: 122 000×, scale bar: 100 nm(FIG. 6E); and chitosan-PEG-TAT/MGF-siRNA nanoparticles, magnification:302 000×, scale bar: 100 nm (FIG. 6F).

FIG. 7 shows mouse neuroblastoma (Neuro2a) cells transfected withmodified chitosan-PEG TAT-siGLO nanoparticles. siGLO (red) is ascrambled siRNA, which is tagged with a Cy3 dye, which upon transfectionindicates the delivery of siRNA.

FIG. 8 shows cytotoxicity study on mouse neuroblastoma cells (Neuro2a)with various treatment using the MTS assay. The results indicate thatnanoparticles with chitosan-PEG-TAT and chitosan-PEG formulation showedminimal toxicity to cells as compared to unmodified chitosannanoparticles. The absorbance was measured at 490 nm using a multiplatecell counter. Control:, Chitosan:, TPP:, siGLO: scrambled siRNA alone,Chitosan-TPP-siGLO:, Chitosan-PEG-TPP-siGLO:,Chitosan-PEG-TAT-TPP-siGLO:.

FIG. 9 shows western blot analysis of ataxin protein performed 24 and 48hours post-transfection. Samples A and C are positive controls withnanoparticles containing no siRNA and scrambled siRNA (siGLO)respectively. Sample B contains nanoparticles with Ataxin1-siRNA.Silencing is observed after 48 hours for sample B. Actin is used as aprotein loading control. The lower panel in this figure provides controlresults for actin.

FIGS. 10A-10B represent histopathological images of brain tissues(cerebral cortex (first row) and cerebellum (second row)) four hoursafter receiving the nanoparticle formulation of biotin-siRNA at variousdoses: 0.25 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg and PBS only (control)(FIG. 10A). The circled regions in column (B), identified with DABstaining, indicate the presence of biotin-tagged scrambled siRNA inneurons observed. Magnification 400×. FIG. 10B graphically illustratesQuantitative analysis of the stained areas of FIG. 10A in tissues usingthe Image J software. Results are shown for the cerebral cortex (darkgray) and the cerebellum (light gray) as percentage of area positive forDAB staining in function of siRNA dose. The graph shows a representativeresult of independent readings from two animals in each group (n=2)mean±s.d. ***P<0.01 was considered highly significant based on Tukey'spost-hoc analysis, when compared with other groups.

FIGS. 11A-11B represent histopathological images (cerebral cortex (firstrow) and cerebellum (second row)) with nanoparticles carrying 0.5 mg/kgof biotin-siRNA at different time points: 4 hrs, 16 hrs, 24 hrs and 48hrs after the administration of the biotin-siRNA (FIG. 11A). The circledregions in column A, identified with DAB staining, indicate the presenceof biotin-tagged scrambled siRNA in neurons delivered by nanoparticles.Magnification 400×. FIG. 11B graphically illustrates Quantitativeanalysis of the stained areas in tissues of FIG. 11A using the Image Jsoftware. Results are shown for the cerebral cortex (dark grey) and thecerebellum (light gray) as percentage of area positive for DAB stainingin function of time elapsed since the administration of thebiotin-siRNA. The graph shows a representative result of independentreadings from two animals in each group (n=2) mean±s.d. ***P<0.01 wasconsidered highly significant based on Tukey's post-hoc analysis, whencompared with other groups.

FIGS. 12A-12B represent biodistribution (through histopathologicalstaining) of various organs collected 4 hrs following administration ofthe nanoparticle formulation containing biotin-siRNA dose at 0.5 mg/kgin animals. In FIG. 12A, results are shown for treated animals (firsttwo columns) and control animals (treated with saline, last two columns)in the cerebral cortex, the cerebellum, the lungs, the heart, the kidneythe liver and the stomach. The results show a maximum delivery ofbiotin-siRNA in the brain (cerebral cortex and cerebellum), with alesser extent in the heart, kidney, liver lungs and stomach.Magnification 400×. FIG. 12B shows quantitative analysis of the stainedareas of FIG. 12A in tissues using the Image J software. Results areshown as percentage of area positive for DAB staining in function oforgan (cerebral cortex, the cerebellum, the lungs, the heart, the kidneythe liver and the stomach) at various doses (from left to right for eachorgan 0.25 mg/kg, 0.5 mg/kg, 1 mg/kg and 2 mg/kg), compared to theuntreated control receiving 0.85% w/v NaCl. The graph shows arepresentative result of independent readings from two animals in eachgroup (n=2) mean±s.d. ***P<0.01 was considered highly significant basedon Tukey's post-hoc analysis, when compared with other groups.

FIGS. 13A-13B represent enlarged DAB stained histopathological sectionsof cerebral cortex (FIG. 13A) and cerebellum (FIG. 13B) withnanoparticle formulation complexing biotin tagged scrambled siRNA atdose 0.5 mg/kg. The cerebral neurons in FIG. 13A and Purkinje cells incerebellum in FIG. 13B show a clear brown staining indicating thepresence and successful delivery of biotin tagged scrambled siRNA inthese cells by nanoparticles administered via intranasal route. Thebrown color obtained by DAB staining in FIGS. 13A and 13B representspresence of biotin-tagged scrambled siRNA in neuronal cells of cerebralcortex and Purkinje cells of cerebellum respectively, as delivered bynanoparticles. Magnification 400×.

FIGS. 14A-14F represent histopathological images of various organtissues collected 4 hrs following intranasal administration ofmultifunctional siRNA/nanoparticle formulation containing biotin-siRNAat a dose of 0.5 mg/kg. The tissues were stained with the TUNELapoptosis assay. Results are shown for the cerebral cortex (FIG. 14A),cerebellum (FIG. 14B), heart (FIG. 14C), lungs (FIG. 14D), kidneys (FIG.14E), liver (FIG. 14F) and stomach (FIG. 14G). As indicated in theseimages, the absence of TUNEL-specific staining represents no apparentcell toxicity/apoptosis in any of the tissues. Magnification 400×.

FIG. 15 shows a schematic diagram of a proposed mechanism of cellularuptake of a nanoparticle device comprising peptide tagged PEGylatedchitosan nanoparticles carrying either nucleic acid (siRNA/DNA/RNA) oran anticancer drug as a therapeutic molecule.

FIG. 16 shows the results associated with the percentage PSN1 geneknockdown upon administration of the nanoparticle composition. Resultsare shown as relative expression (in function of GADPH) of PSN1 foranimals treated with the nanoparticles bearing a PSN1 siRNA (leftcolumn) and for control (untreated) animals (right column). The resultsindicate a 21.34% reduction in PSN1 gene expression with nanoparticlescarrying siRNA against PSN1 gene, when compared with untreated control.The graph shows a representative result (average of n=4±S.E.).

FIGS. 17A-17E show the result of serum analysis performed on animalstreated with the nanoparticles bearing the PSN1 siRNA or a control.Results are shown for ALP levels (U/L) (FIG. 17A), AST/ALT levels (U/L)(FIG. 17B), CRP-5 (mg/L) (FIG. 17C), urea (mmol/L) (FIG. 17D), CRE(μmol/L) (FIG. 17E) and UA (μmol/L) (FIG. 17F) in function of treatment.The graphs show representative results (average of n=4±S.E.). *P<0.05was considered significant based on student t-test.

FIG. 18 shows ¹H NMR spectra of Chitosan-PEG-TAT (CS-O-PEG-CONH-TAT)(plot A), Chitosan-PEG-MGF (CS-O-PEG-CONH-MGF) (plot B),Chitosan-PEG-P160 (CS-O-PEG-CONH-P160) (plot C) and Chitosan-PEG-CP15(CS-O-PEG-CONH-CP15) (plot D). The multiple peaks of oxymethyl groups inPEG at δ 3.3 to 3.7 cover over the signals of pyranose ring of chitosan.The multiple peaks at δ 6.0-9.0 belong to the peptide sequencesrespectively.

FIG. 19 shows transmission electron microscopy (TEM) image ofpeptide-tagged pegylated chitosan nanoparticles complexing siRNA.Magnification at 95 800×. Scale bar: 500 nm. The average size of thenanoparticles ranged from 100 to 200 nm.

FIG. 20 shows gel retardation assay results to evaluate the maximum gene(siRNA) loading efficiency of nanoparticles. Lane 1 represents a 10 bpDNA ladder used as a reference. Lane 2 represents 2 μg/ml, lane 3represents 4 μg/ml, lane 4 represents 6 μg/ml, lane 5 represents 8 μg/mland lane 6 represents 10 μg/ml of siRNA dose complexed with thenanoparticles. The results suggest that 8 μg/ml is the optimal amount ofsiRNA to be complexed with the nanoparticles.

FIGS. 21A-21E represent results of nanoparticles accumulation in thetumor tissue after 4 hrs of dose administration. FIGS. 21A-D show imagesof SW480 colon cancer indicate the presence of scrambled biotinylatedsiRNA (0.5 mg/kg) in the tumor tissue after intraperitonealadministration of the treatment nanoparticle formulation. Animals weresacrificed after 4 hrs. Results are shown for the following nanoparticleformulations: chitosan-PEG-CP15 (FIG. 21A), unmodified chitosannanoparticles (FIG. 21B), non-targeted biotin siRNA alone (FIG. 21C) anduntreated control cells (FIG. 21D). FIG. 21E shows image analysis of themean percent area stained in the tumor tissues of FIGS. 21A-21D. Resultsare shown as a percentage of biotin-NT siRNA expression in function oftreatment (chitosan-PEG-CP15: black bar, unmodified chitosannanoparticles: light gray bar, non-targeted biotin siRNA alone: darkgray bar and untreated control cells. The graph shows a representativeresult of the average of three random sections measured per animaltissue, mean±s.d. *p<0.05, **p<0.01 were considered significant based onTukey's post-hoc analysis, when compared with other groups.

FIGS. 22A-22B represent analysis of biodistribution of siRNA in varioustissues 4 hrs after the administration of various nanoparticlesformulations. FIG. 22A shows histopathological staining of heart, lungs,kidney, liver and spleen obtained from a mouse xenografted with coloncancer and treated with various nanoparticle formulations (administeredat a dose of 0.5 mg/kg) CS-P-CP15-NT siRNA: chitosan-PEG-CP15, CS-NTsiRNA: unmodified chitosan nanoparticles, NT-siRNA: non-targetingbiotin-siRNA alone and control as untreated. FIG. 22B shows imageanalysis of the mean percent area stained in the tumor tissues shown inFIG. 22A. Results are shown as mean percentage area stained for thepresence of the siRNA in function of the tissue studied and thenanoparticle formulations administered (from left to right for eachorgans, CS-P-CP15 NT siRNA, CS-NT siRNA, NT siRNA and control). Thegraphs presents representative results of the average of two randomsections measured per animal tissue, mean±s.d. *p<0.05, **p<0.01 wereconsidered significant based on Tukey's post-hoc analysis, when comparedwith other groups.

FIG. 23 shows PLK1 mRNA expression in the tumor tissue afterintra-peritoneal administration with various nanoparticle formulations.Results are shown as relative gene expression of PLK1 in tumor tissuesin function of nanoparticle formulation: CS-PEG-CP15-siRNA (●),CS-PEG-CP15 NT siRNA with non-targeting biotin-siRNA (▪), PLK1 siRNAalone (▴) and saline (control ▾). The PLK1 gene expression was comparedamong different groups after normalizing the GAPDH levels among all theanimals. A 50% PLK1 gene suppression was observed in treated animals(p=0.031) as compared with untreated controls. The graph shows a scatterdot plot with n=6 and mean±SE, *p<0.05 was considered significant basedon Tukey's post-hoc analysis, when compared with other groups.

FIG. 24 shows PLK1 protein expression in the tumor tissue afterintra-peritoneal administration with various nanoparticle formulations.Results are shown as relative PLK1 protein expression (obtained bywestern blot analysis from 100 μg of total protein extracted from tumortissues of colon cancer and normalized against the β-actin proteinlevels) in function of nanoparticle formulations: CS-PEG-CP15 with siRNAagainst PLK1 gene (●), CS-PEG-CP15 with non-targeting biotin-siRNA (▪),PLK1 siRNA alone (▴) and saline (control ▾). Reduction in PLK1 proteinexpression was observed with treatment formulation as compared withuntreated and controls. The graph shows a scatter dot plot with n=6 andmean±SE, *P<0.05 was considered significant based on Tukey's post-hocanalysis, when compared with other groups.

FIGS. 25A-25B show results of serum analysis of mouse treated withvarious nanoparticle formulations. In FIG. 25A, AST/ALT ratios (U/L) areshown for mouse treated with CS-PEG-CP15 with siRNA against PLK1 gene(●), CS-PEG-CP15 with non-targeting biotin-siRNA (▪), PLK1 siRNA alone(▴) and saline (control ▾). In FIG. 25B, C-reactive protein levels(mg/dl) are shown for mouse treated with CS-PEG-CP15 with siRNA againstPLK1 gene (●), CS-PEG-CP15 with non-targeting biotin-siRNA (▪), PLK1siRNA alone (▴) and saline (control ▾). No significant differencesbetween the treatment group and untreated controls were observed. Thegraph shows a scatter dot plot with n=5 and mean±SE.

FIGS. 26A-26B show a CT scan of the neck before (FIG. 26A) and after(FIG. 26B) the 10 day therapy cycle of Nora-PLK1 therapy (intra-tumor),initiated on Sep. 27, 2014. The CT obtained on Oct. 13, 2014 shows adramatic reduction of the tumor mass. Volumetrically the tumor reducedfrom 29.66×41.66×46 mm to 18.66×35.55×41 mm or from 56,839.2 m³ to27,197.8 m³ (a 46% reduction).

FIGS. 27A-27D show photographic evidence of the reduction of stage IV,squamous cell carcinoma (head and neck cancer) with the Nora-PLK1therapy (nanoparticle-siRNA), administered directly into the tumor of a55 years old, female patient. The dose administered was 100 μg ofsiRNA/day. FIG. 27A represents the state of tumor, prior to therapy.FIG. 27B represents, reduction in the size of tumor within first 3 weeksof the therapy. FIG. 27C represents, complete reduction in the size ofthe tumor with presence of only scar tissue. FIG. 27D represents,complete disappearance of tumor mass and scarring tissue from the neck.

FIGS. 28A-28B show a chest CT scan of a 55 year old female patient. FIG.28A represents the tomogram from the Chest CT from Sep. 27, 2014 andFIG. 28B represents the Chest x-ray from Nov. 26, 2014. As observed, atumor mass is seen on the upper part of the lower lobe of the right lungin FIG. 28A (white-dotted circle) that is not present on the X-ray ofFIG. 28B.

DETAILED DESCRIPTION

The present invention provides a delivery system for specificallydelivering agents to a tissue (or a group of tissues), an organ (or agroup of organs) or a cell type (or a group of cell types). The deliverysystem is referred to as a non-viral nanoparticles composed of aplurality of polymeric materials, each comprising a hydrophilic linearpolymer (such as PEG) covalently associated at one end with across-linkable cationic polymer and at the other end with atargeting/penetrating peptide. The nanoparticles are not based on aviral system for delivering agents. The cellular specificity of thedelivery system can be used to lower the dose of the agent used and,consequently, limit, if any, the undesirable effects associated with theagent. The nanoparticles can be used in vitro for research purposes, exvivo for cellular therapy applications as well as in vivo for therapy ordiagnostics. In some embodiments, the nanoparticles can be designed tobe more efficient at translocating across a biological membrane and/or acellular membrane (such as, for example, the cytoplasmic membrane or thenuclear membrane).

Polymeric Material for Non-Viral Nanoparticles

The present invention provides a polymeric material that can be used forthe production of non-viral nanoparticles. The polymeric materialcomprises at least three components, a linear hydrophilic polymer(preferably a linear polyethylene glycol polymer), a cross-linkablecationic polymer (preferably a chitosan polymer) and atargeting/penetrating peptide. In the polymeric material, the linearhydrophilic polymer is covalently associated, at one end, to thecross-linkable cationic polymer and, at the other end, to thetargeting/penetrating peptide. The targeting/penetrating peptide isindirectly covalently associated with the cationic cross-linkablepolymer (through the linear hydrophilic polymer) but is not directlycovalently associated with the cross-linkable cationic polymer.

The components of the polymeric material can be directly and covalentlyassociated with one another. In such embodiment, there is no linkerbetween the cross-linkable cationic polymer and the linear hydrophilicpolymer nor between the linear hydrophilic polymer and thetargeting/penetrating peptide. However, in an alternative embodiment, insome applications, a linker (either between the cross-linkable cationicpolymer and the linear hydrophilic polymer and/or between the linearhydrophilic polymer and the targeting/penetrating peptide) can bepresent. In some instances, the linker can only be present between thecross-linkable cationic polymer and the linear hydrophilic polymer orbetween the linear hydrophilic polymer and the targeting/penetratingpeptide. In other instances, the linker can be present between thecross-linkable cationic polymer and the linear hydrophilic polymer andbetween the linear hydrophilic polymer and the targeting/penetratingpeptide. The linker between the cross-linkable cationic polymer and thelinear hydrophilic polymer can be the same or can differ from the linkerbetween the linear hydrophilic polymer and the targeting/penetratingpeptide.

The cross-linkable cationic polymer of the polymer biomaterial can becross-linked to provide an internal core of cross-linked cationicpolymer. In addition, because of the positive charge of thecross-linkable polymer, the internal core provided upon cross-linkingprovides a micro-environment for a non-covalent association with ananionic agent. In some embodiments, the cross-linking between differentpolymeric material only occurs between the cationic polymer moieties offrom at least two polymeric material. Otherwise stated, no or verylittle cross-linking occurs between the cationic polymer and the linearhydrophilic polymer or the targeting/penetrating peptide, no or verylittle cross-linking occurs between the linear hydrophilic polymermoieties of at least two distinct polymeric material, no or very littlecross-linking occurs between the linear hydrophilic polymer and thetargeting/penetrating peptide, and/or no or very little cross-linkingoccurs between the targeting/penetrating peptide moieties of at leasttwo distinct polymeric material.

In a preferred embodiment, the cross-linkable cationic polymer ischitosan. In some embodiments, chitosan can be modified through itsamine groups. However conjugating other compounds through amines ofchitosan can lead to the loss of its cationic nature. The presence offree amine groups (e.g. a net positive charge) on chitosan can help incomplexing with negatively charged agents (due to ionic interactions),can help in cellular uptake and, once inside the endosomal cavity, cancreate a “proton sponge effect” (e.g. a swelling behavior of chitosanobserved when encountering an acidic pH inside the cell's endosome).However, it was observed that these free amine groups can be responsiblefor a mild cellular toxicity. In the polymeric material describedherein, chitosan was chemically modified in order to preserve some ofits inherent cationic nature. In some embodiments, the amine groups ofthe chitosan cause a surface charge (of the resulting nanoparticlepreferably having a size of less than about 100 nm) between 15 to 25 mV.In order to do so, the amine groups can be first be protected then thechitosan can be conjugated the other functional groups on chitosan withthe hydrophilic linear polymer. The expression “protecting amine groups”refer to the protection an amine moiety with any suitable protectinggroups. Examples of amine protecting group can be found in Green et al.,“Protective Groups in Organic Chemistry”, (Wiley, 4^(th) ed. 2007) andHarrison et al. “Compendium of Synthetic Organic Methods” (John Wileyand Sons, 1996).

In the polymeric material described herein, the degree of deacetylationof the chitosan used can be 70%, 75%, 80%, 85% or 90%. In someembodiment, the degree of deacetylation of chitosan can be 80%. Inaddition, in some embodiments, the chitosan used can be a low (10-50KDa), medium (50-200 KDa) or high (200-500 KDa) molecular weightchitosan. In preferred embodiments, the chitosan has an averagemolecular weight of the chitosan can be, for example, about 100 000 Da.

Chitosan is unique among polysaccharides because it carries amino groupswhich are positively charged in mildly acidic aqueous solution (pH≤5.5).As indicated above, the amine groups can also be coupled to variouschemical groups. In the polymeric material described herein, thechitosan is modified (e.g. covalently modified) to be attached (e.g.directly attached) through a hydroxyl group to the linear hydrophilicpolymer. In some embodiments, various chitosan molecules also can becross-linked together via their amino groups.

In some embodiments, chitosan is not a water soluble variety ofchitosan. Water soluble chitosan are usually polymeric oligomers, whichare not efficient in complexing nucleic acid molecules, such as siRNA.Due to the small size of the water-soluble polymeric oligomers and thesiRNA, the complexes formed were shown not to be stable and were readilydegraded by the serum proteins. As such, if a water soluble chitosan isused, it must be able to form a stable complex with the anionic agent,such complex being capable of resisting degradation from serum proteins.

In another preferred embodiment of the polymeric material, the linearhydrophilic polymer can be a linear polyethylene glycol (PEG) polymer.Throughout this application, the action of attaching a linear PEGpolymer to another entity is referred to as “PEGylation” or “PEGylating”and a material having been attached to a linear PEG can be qualified as“PEGylated”. Once covalently associated with the chitosan molecule, thePEG can reduce the steric-hindrance of the nano-particle formed with thepolymeric material as well as reduce the inherent mild cellularcytotoxicity associated with non-PEGylated chitosan. In preferredembodiments, the PEG has an average molecular weight between about 2000Da. In some other specific embodiments, the PEG can also have an averagemolecular weight of about 1 000 to 5 000 Da.

The third component of the polymeric material is a targeting/penetratingpeptide. As indicated herein, the targeting/penetrating peptide has theability to specifically bind to a cell surface or to a cellularcomponents. It thus provides cell specificity and can also increasecellular uptake of the polymeric material (as well as the non-viralparticle produced from the polymeric material). It can, for example,facilitate the crossing of a cellular membrane (such as, for example,the nuclear membrane). Alternatively, the targeting/penetrating peptidecan be to a cell surface or cell-associated surface receptor. In suchembodiments, the targeting/penetrating peptide can be a fragment orderivative of a protein transduction domain or a fragment or derivativeof a growth factor. In this application, a single targeting/penetratingpeptide is covalently associated per linear hydrophilic polymer.However, this does not limit the use single peptide per nanoparticleformulation. For example, combination of peptides can be used during thesynthesis of the polymeric material for making a population of differentpolymeric material, each bearing a single peptide that can differamongst the members of the population. The use of a population ofpolymeric material can further be used for the production of amultifunctional non-viral nanoparticle.

In some embodiments, the targeting/penetrating peptide can facilitatethe translocation through a biological membrane. Suchtargeting/penetrating peptide can be a TAT oligopeptide covalentlyconjugated to the PEG polymer. TAT is a transcriptional activatorprotein encoded by human immunodeficiency virus type 1 (HIV-1) and isinvolved in the replication of the HIV virus. The basic domain of TATpeptide comprises mainly of arginine and lysine amino acid residues thathave shown to play an important role in translocation across biologicalmembrane due to its strong cell-adherence and is independent ofreceptors, temperature. The TAT peptide can be used for facilitating thetranslocation of the nanoparticles across any biological membrane.Nanoparticles comprising polymeric material bearing the TAT peptide canalso contain other polymeric material bearing othertargeting/penetrating peptides.

In other embodiments, the targeting/penetrating peptide can be specificfor neuronal cell. Such target peptide include, but are not limited toMGF. MGF is a mechano growth factor. It is a splice variant of IGF-1(Insulin Growth Factor-1), whose receptors are abundantly present on thesurface of Purkinje cells in the cerebellum. IGF-1 is a growth-promotingfactor for Purkinje cell development, particularly post-natal survivaland dendritic growth. IGF-1 is majorly involved in motor learningprocess and purkinje cell synaptic plasticity. MGF has shown to promotemotor neuron survival but its neuroprotective action is independent ofIGF-1. MGF has been identified to have an affinity for myocardial andneurological tissues and can be used as an effectivetargeting/penetrating peptide for these tissues.

As also indicated herein, in some embodiments, the targeting/penetratingpeptide can be CP15. CP15 was identified by the phage displaytechnology. CP15 peptide has shown to be the most effective peptidetargeting colon tumor cells but not the normal human intestinalepithelial human cells. CP15 peptide is used as a ligand that will guidethe nanoparticles to selectively target the tumor tissue expressingreceptors for CP15 peptide (colon cancer).

In some embodiments, the targeting/penetrating peptide can be P160. P160was identified by phage display technology. P160 peptide has shown highaffinity towards breast cancer and neuroblastoma cells without affectingthe normal endothelial cells. P160 can be used as a ligand that willguide the nanoparticles to selectively target the tumor tissueexpressing receptors for P160 peptide (breast cancer).

Process for Producing the Polymeric Material

In order to produce the polymeric material, the hydrophilic linearpolymer can be first covalently associated with the cross-linkablecationic polymer. Such covalent binding can be a direct binding in whichno linker is placed between the cross-linkable cationic polymer and thehydrophilic linear polymer. Alternatively, such covalent binding can bean indirect binding where a linker is placed between the cross-linkablecationic polymer and the hydrophilic linear polymer. Once thehydrophilic linear polymer is covalently associated with thecross-linkable cationic polymer, then the hydrophilic polymer iscovalently associated with the targeting/penetrating peptide. Suchcovalent binding can be a direct binding in which no linker is placedbetween the targeting/penetrating peptide and the hydrophilic linearpolymer. Alternatively, such covalent binding can be an indirect bindingwhere a linker is placed between the targeting/penetrating peptide andthe hydrophilic linear polymer. As indicated above, there is no directcovalent association or binding between the targeting/penetratingpeptide and the cross-linkable cationic polymer (even though somenon-covalent association may nevertheless be observed between thetargeting/penetrating peptide and the cross-linkable cationic polymer).

In embodiments where the cross-linkable cationic polymer is chitosan,some of (in some embodiments, the majority of) the amine groups of thechitosan molecule can be protected prior to its covalent associationwith the linear hydrophilic polymer. Such protection can be conferred,for example, by phthaloylation. In such embodiment, the amine groups ofthe chitosan molecule can be de-protected after the covalent associationof between the hydrophilic linear polymer and the targeting/penetratingpeptide.

In some embodiments, a derivative of the linear hydrophilic polymer canbe used in the process. When the linear hydrophilic polymer is PEG, oneof the derivatives that can be used is monomethyl ether polyethyleneglycol (mPEG-OH). In such embodiment, the mPEG can first be reacted(with succinic anhydride for example) to form mPEG-COOH and then reacted(with thionyl chloride for example) to form a mPEG-COCl. The mPEG-COCl(having a reactive first end) can then be reacted with thecross-linkable cationic polymer to form a PEGylated cross-linkablecationic polymer. Once the PEGylated cross-linkable cationic polymer hasbeen formed, the other end of PEG containing methylether group (—OCH₃)can also be reacted to convert it to a hydroxyl group (with aluminiumchloride in ethanethiol for example) and then reacted to form anactivated PEG-COOH (with succinic anhydride for example). The activatedPEG-COOH (having a reactive second end) can then be reacted with the atleast one targeting/penetrating peptide to form the complete polymericmaterial.

In an alternate embodiment to produce the polymeric material, the PEGcan be first covalently associated with the targeting/penetratingpeptide and then with the cross-linkable cationic polymer. In suchembodiment, the mPEG can first be reacted (with succinic anhydride forexample) to form mPEG-COOH and then reacted (with thionyl chloride forexample) to form a mPEG-COCl. The mPEG-COCl (having a reactive firstend) can then be reacted with the targeting/penetrating peptide to forma PEGylated targeting/penetrating peptide. Once the PEGylatedtargeting/penetrating peptide has been formed, in an additionalembodiment, monomethyl ether polyethylene glycol (mPEG-OH) can also bereacted to convert one of its methyl ether group to a hydroxyl group(with aluminium chloride in ethanethiol for example) and then reacted toform an activated PEG-COOH (with succinic anhydride for example). Theactivated PEG-COOH (having a reactive second end) can then be reactedwith the cross-linkable cationic polymer to form the polymeric material.

The person of ordinary skill in the art will understand that the processdescribed herein can be optimized and modified to accommodate thevarious applications of the polymeric material and should not beintended to be use to limit the scope of the invention.

Non-Viral Nanoparticles

Due to the nature of the polymeric material used, the nanoparticlesdescribed herein can be formed by simply admixing an anionic agent withthe positively charged cross-linkable polymer. Through ionicinteractions, the anionic agent complexes with the cationiccross-linkable polymer. Upon the addition of a cross-linker, the anionicagent becomes entrapped in the cross-linked internal core formed by thecationic polymer. In the non-viral nanoparticles, the anionic agent doesnot form a distinct structure but is within the cross-linked mesh of thecationic polymer. In some embodiments, the non-viral nanoparticles areshown to exhibit low in vitro cytotoxicity. In embodiments, thenanoparticles have been shown to cross physiological barrier such as theblood-brain barrier (for example, when they have been administeredintra-nasally). In other embodiments, the nanoparticles have been shownto be specifically delivered to a tissue (for example, a solid tumor).In some embodiments, the nanoparticles which comprise nucleic acidmolecules as the entrapped anionic agent have efficiently released thenucleic acid into the cell's cytoplasm. In additional embodiments, thenanoparticles can be designed for being targeted to the cell nucleus (byusing, for example, as one of the targeting/penetrating peptide, apeptide that can facilitate the transfer through the nuclear membrane).

As disclosed herein, a surface functionalized biocompatible andbiodegradable chitosan nanoparticles was developed and can be used toincrease the efficacy and stability of the nucleic acid-basedtherapeutics and/or other therapeutic molecules. In embodiments, thenanoparticles have been shown to provide sustained targeted delivery.Without wishing to be bound to theory, FIG. 15 shows a schematic diagramof a proposed cellular uptake mechanism of the non-viral nanoparticles.In this figure, the nanoparticles are able to release the anionic agentat its targeted site. A targeting ligand can be a specific peptidetargeting, for example, a cell-specific receptor. The use of atargeting/penetrating peptide avoids the invasive delivery strategiessuch as implantation of catheters, intracarotid infusions fortherapeutic delivery, surgeries and chemotherapies.

The nanoparticles-based drug-delivery system provided herein isrelatively inert and has low integration with physiological systembefore reaching its targeted cell, organ or tissue. Some of thenanoparticles can handle more payload, be modified with appropriateligands for specific cell targeting and be administrated repeatedlywithout fretting about delivery induced toxicity or immunogenicity. Someof the nanoparticles can be designed for being less than about 100 nm indiameter and have a positive surface charge. Some of the nanoparticlescan overcome anatomical, biophysical and physiological barriers, such asthe blood brain barrier. Some of the nanoparticles are able tosafe-guard the anionic agent against degradative enzymes before reachingthe targeted site. Some of the nanoparticles can be designed to betarget specific and/or can be administered non-invasively.

In order to design the non-viral nanoparticles, various properties ofits constituents have been exploited. As indicated above, one embodimentof the cross-linkable cationic polymer is chitosan. Chitosan is apolycationic polymer that has been regarded as a non-toxic,biodegradable and a biocompatible polysaccharide. Due to the presence ofprimary amine groups, it is only soluble in dilute acids. However, thesolubility can be controlled by specifically modifying the primary aminegroups thereby making it soluble in solvents like DMSO, DMF, pyridine,THF, etc. Chitosan of low molecular weight has been shown to complexnucleic acids like DNA at acidic pH and exhibit complex stability andintegrity.

In addition, chitosan is also considered to be a mucoadhesive agentwhich can reduce the clearance rate from the nasal cavity. As such, ininstances where the nanoparticles are formulated for intranasal delivery(for example in intranasal drops), it is believed that the nanoparticlesare absorbed across the nasal epithelia tissue, i.e. olfactoryepithelium, and follow olfactory/trigeminal neural pathways. In suchembodiment, the pH at which the nanoparticles can be formulated is 6.0,which is in accordance with the pH range maintained in nasal cavity (pH5.0 to 6.5). Chitosan has also been shown to interact with sialic acidresidues in the mucus to open the tight endothelial junctions. It hasfurther been shown to be used for intranasal delivery of proteins.

As also indicated above, in some embodiments, the linear hydrophilicpolymer is polyethylene glycol (PEG). This polymer has been extensivelyused in biomedical and pharmaceutical applications because of itshydrophilic, non-toxic, non-antigenic and non-immunogenic features. Itis also a polymer which can be chemically modified easily. Inparticularly, the use of monofunctional PEG avoids nanoparticles toagglomerate and provides resistance against enzyme degradation. In thecontext of the present invention, the use of a bifunctional PEG (such asmPEG-COOH) serves as a linker for conjugating two different entities (across-linkable cationic polymer and a targeting/penetrating peptide).Because it is neutral in charge and reduces the steric-hindrance of thecharged polymers, PEG enables efficient conjugation of the two entitieslinked on its either side.

The non-viral nanoparticles can comprise a single type of polymericmaterial bearing the same linear hydrophilic polymer, the samecross-linkable cationic polymer and the same targeting/penetratingpeptide. Alternatively, the non-viral nanoparticles can also be made upof different polymeric materials. For example, the non-viralnanoparticles can be made up of polymeric material having the samelinear hydrophilic polymer but different cross-linkable cationicpolymers and different targeting/penetrating peptides. In anotherexample, the non-viral nanoparticles can be made up of polymericmaterial having the same linear hydrophilic polymer and the samecross-linkable cationic polymer but different targeting/penetratingpeptides. In yet another example, the non-viral nanoparticles can bemade up of polymeric material having different linear hydrophilicpolymers but the same cross-linkable cationic polymer and the sametargeting/penetrating peptide. In still another example, the non-viralnanoparticles can be made up of polymeric material having the samelinear hydrophilic polymer and the same different targeting/penetratingpeptide but different cross-linkable cationic polymers. In yet anotherexample, the non-viral nanoparticles can be made up of polymericmaterial having the same cross-linkable cationic polymer but differentlinear hydrophilic polymers and different targeting/penetratingpeptides. In still another example, the non-viral nanoparticles can bemade up of polymeric material having the same targeting/penetratingpeptide, but different linear hydrophilic polymers and differentcross-linkable cationic polymers.

In a specific embodiment, the non-viral nanoparticle is composed of aplurality of polymeric materials. One of the polymeric material cancomprise a first polymeric material having a first targeting/penetratingpeptide that can bind to a cell surface or cell-associated receptor (forexample, the MGF, CP15 or P160 targeting/penetrating peptide). Anotherpolymeric material can comprise a second polymeric material having asecond peptide that can facilitate the translocation of the non-viralparticle across a cellular membrane (for example the TAT penetratingpeptide). Such nanoparticles is thus composed of at least two differenttargeting/penetrating peptides, one for facilitating the translocationacross a biological membrane and another one for providing cellular ortissue-specificity.

The non-viral nanoparticles comprise an anionic agent. The agent shouldbear a net negative charge in order to complex easily with the cationicpolymer. The agent can be used for research purposes, therapeuticpurposes and/or diagnostic purposes.

In some embodiment, the anionic agent is a nucleotide-based agent forlowering or inhibiting gene expression and/or protein expression. Suchagents include, but are not limited to antisense oligonucleotide,triplex oligonucleotide, miRNA, siRNA, shRNA and ribozyme. In someembodiments, a single type of anionic agent is present in the non-viralnanoparticles. In other embodiments, more than one types of anionicagent is present in the non-viral nanoparticles.

An antisense oligonucleotide is wholly or partially complementary to,and can hybridize with, a target nucleic acid (either DNA or RNA). Forexample, an antisense nucleic acid or oligonucleotide comprising about15 to 35 nucleotides spanning the coding/non-coding sequence of a geneor its corresponding transcript whose expression is to be inhibited. Inanother embodiment, the antisense nucleic acid is wholly or partiallycomplementary to, and can hybridize with, a target nucleic acid. Asnon-limiting examples, antisense oligonucleotides may be targeted tohybridize to the following regions: mRNA cap region, translationinitiation site, translational termination site, transcriptioninitiation site, transcription termination site, polyadenylation signal,3′ untranslated region, 5′ untranslated region, 5′ coding region,mid-coding region, 3′ coding region, DNA replication initiation andelongation sites.

Triplex oligonucleotides, much like antisense oligonucleotides caninhibit transcription and/or expression of a target gene or itstranscripts. Triplex oligonucleotides are constructed using thebase-pairing rules of triple helix formation and the nucleotide sequenceof the target genes. They are usually between 10 and 40 or between 15 to25 nucleotides-long.

RNA interference (RNAi) is a post-transcriptional gene silencing processthat is induced by a miRNA or a dsRNA (a small interfering RNA (siRNA)or small hairpin RNA (shRNA)) and has been used to modulate geneexpression. While the invention is not limited to a particular mode ofaction, RNAi may involve degradation of messenger RNA by an RNA inducedsilencing complex (RISC), preventing translation of the transcribedtargeted mRNA. Oligonucleotides that can mediate RNAi are generally atleast 10 nucleotides long. Because manipulation of RNA may be complex,RNAi can be provided with a deoxyribonucleic acid (DNA) compositionsencoding small interfering RNA (siRNA) molecules, or intermediate siRNAmolecules (such as shRNA), comprising one strand of an siRNA.

Small interfering RNA or siRNA includes any nucleic acid moleculecapable of mediating RNA interference “RNAi” or gene silencing. Forexample, siRNA of the present invention can be double stranded RNAmolecules from about 10 to about 30 nucleotides long that are named fortheir ability to specifically interfere with protein expression. In oneembodiment, siRNAs of the present invention are 12 to 28 nucleotideslong, more preferably 15 to 25 nucleotides long, even more preferably 19to 23 nucleotides long and most preferably 21 to 23 nucleotides long.Therefore preferred siRNA of the present invention are 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 nucleotides inlength. As used herein, siRNA molecules need not to be limited to thosemolecules containing only RNA, but further encompass chemically modifiednucleotides and non-nucleotides. An siRNA molecule can be assembled fromtwo nucleic acid fragments wherein one fragment comprises the senseregion and the second fragment comprises the antisense region of siRNAmolecule. The sense region and antisense region can also be covalentlyconnected via a linker molecule. The linker molecule can be apolynucleotide linker or a non-polynucleotide linker. In an embodiment,the siRNA can target the PSN1 gene or the PLK1 gene.

A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme orcatalytic RNA) is an RNA molecule that catalyzes a chemical reaction.Some ribozymes may play an important role as therapeutic agents, asenzymes which target defined RNA sequences. Ribozymes can be geneticallyengineered to specifically cleave a transcript of a gene from acandidate region that is being upregulated with the disease.

The oligonucleotides described herein can be naturally-occurring speciesor synthetic species formed from naturally-occurring subunits or theirclose homologs. The term may also refer to moieties that functionsimilarly to oligonucleotides, but have non-naturally-occurringportions. Thus, oligonucleotides may have altered sugar moieties orinter-sugar linkages. Exemplary among these are phosphorothioate andother sulfur containing species which are known in the art. In someembodiments, at least one of the phosphodiester bonds of theoligonucleotide has been substituted with a structure that functions toenhance the ability of the compositions to penetrate into the region ofcells where the RNA whose activity is to be modulated is located. It ispreferred that such substitutions comprise phosphorothioate bonds,methyl phosphonate bonds, or short chain alkyl or cycloalkyl structures.In accordance with other embodiments, the phosphodiester bonds aresubstituted with structures which are, at once, substantially non-ionicand non-chiral, or with structures which are chiral and enantiomericallyspecific. Persons of ordinary skill in the art will be able to selectother linkages for use in the practice of the invention.Oligonucleotides may also include species that include at least somemodified base forms. Thus, purines and pyrimidines other than thosenormally found in nature may be so employed. Similarly, modifications onthe furanosyl portions of the nucleotide subunits may also be affected,as long as the essential tenets of this invention are adhered to.Examples of such modifications are 2′-O-alkyl- and2′-halogen-substituted nucleotides. Some non-limiting examples ofmodifications at the 2′ position of sugar moieties which are useful inthe present invention include OH, SH, SCH₃, F, OCH₃, OCN, O(CH₂), NH₂and O(CH₂)_(n)CH₃, where n is from 1 to about 10. Such oligonucleotidesare functionally interchangeable with natural oligonucleotides orsynthesized oligonucleotides, which have one or more differences fromthe natural structure.

In other embodiments, the anionic agent is a nucleotide-based agent forpromoting or increasing gene expression and/or protein expression. Suchagents include, but are not limited to expression vectors. In someembodiments, a single type of anionic agent is present in the non-viralnanoparticles. In other embodiments, more than one types of anionicagent is present in the non-viral nanoparticles.

Expression vectors can be derived from viruses (retroviruses,adenovirus, adeno-associated viruses, herpes, lentiviruses and/orvaccinia viruses) or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express nucleic acidsequence that is complementary to a specific (or a combination) ofnucleic acid sequence. Viral vector delivery systems include DNA and RNAviruses, which have either episomal or integrated genomes after deliveryto the cell.

The non-viral nanoparticles of the invention are small enough totranslocate across a biological membrane. In some embodiment thediameter of the nanoparticles is between about 5 to about 100 nm. Inother embodiments, the diameter of the nanoparticles are between about50 to about 300 nm. In an embodiment, the non-viral nanoparticles have adiameter of less than 5 nm. In another embodiment, the non-viralnanoparticles have a diameter between 5 and 10 nm. In a furtherembodiment, the non-viral nanoparticles have a diameter between 100 and200 nm.

Process for the Production of the Non-Viral Nanoparticles

In some embodiments, the non-viral nanoparticles can be obtained in aone-step fashion. A solution comprising both the anionic agent and thecross-linker is added to a plurality of polymeric materials (identicalor different). In an embodiment, when the anionic agent is a siRNA, theratio between the siRNA and the polymeric materials is at least 2 μg/ml,at least 4 μg/ml, at least 6 μg/ml, at least 8 μg/ml or at least 10μg/ml of the siRNA for each 0.5 mg/ml of the polymeric materialsolution. In a preferred embodiment, the ratio between the siRNA and thepolymeric materials is about 2 μg/ml of siRNA to be complexed 0.5 mg/mlof polymeric material solution (with average molecular weight ofchitosan of 10 KDa) to form nanoparticles in range 5 to 100 nm. Inanother preferred embodiment, the ratio between the siRNA and thepolymeric material is about 8 μg/ml of the siRNA for each 0.5 mg/ml ofthe polymeric material solution (with average molecular weight ofchitosan 50 to 190 KDa) to form nanoparticles in range 50 to 300 nm. Theprocess for making a non-viral nanoparticle includes an anionic crosslinker, that can be sodium tripolyphosphate (TPP). The anionic agent(siRNA) along with the anionic cross-linker (TPP) are admixed and thenadded drop-wise to a cationic polymeric solution in conditions favoringthe formation of nanoparticles through ionic interactions. As indicatedherein, cross-linking is limited to the cross-linkable cationic polymermoiety of the polymeric material. Cross-linking is not observed betweenthe other components of the polymeric material or the anionic agent. Theresulting nanoparticles will have an internal core (in which the anionicagent is entrapped in the cross-linked cationic polymer) havingprotrusions of hydrophilic linear polymers being covalently associatedwith the targeting/penetrating peptide. The size of the resultingnanoparticles are in the nanometers range. The shape of the resultingnanoparticles is spherical. In some embodiments, the relative diameterof the non-viral nanoparticle is between about 5 to about 100 nm. Inother embodiments, the relative diameter of the non-viral nanoparticleis between about 50 to about 300 nm.

Exemplary cross-linkers include, but are not limited to small chemicalentities having either amine reactive groups, carboxyl reactive groups,aldehyde reactive groups, ketone reactive groups, hydroxyl reactivegroups, thiol reactive groups and hydrazide reactive groups. Examples oflinkers can be found in Greg T. Hermanson, “Bioconjugate techniques”(Elsevier Inc. 2008). More specifically, the cross-linkers include, butare not limited to zero-length crosslinkers (such as, for example,carbodiimides (EDC, EDC and sulfo-NHS, CMC, DCC, DIC), Woodward'sReagent K, N, N-carbonyldiimidazole and Schiff Base Formation andReductive Amination), homobifunctional crosslinkers (such as, forexample, homobifunctional NHS esters (DSP and DTSSP, DSS and BS, DST andSulfo-DST, BSOCOES and Sulfo-BSOCOES, EGS and Sulfo-EGS, DSG, DSC),homobifunctional imidoesters (such as, for example DMA, DMP, DMS, DTBP),homobifunctional sulfhydryl-reactive crosslinkers (DPDPB, BMH,difluorobenzene derivatives, DFDNB, DFDNPS), homobifunctionalphotoreactive crosslinkers (such as, for example, BASED),homobifunctional aldehydes (such as, for example, formaldehyde,glutaraldehyde), bis-epoxides (such as, for example, 1,4-butanedioldiglycidyl ether), homobifunctional hydrazides (such as, for example,adipic acid dihydrazide), carbohydrazide (such as, for example,bis-diazonium derivatives, o-Tolidine, diazotized, bis-diazotizedbenzidine) and bis-alkyl halides), heterobifunctional crosslinkers(amine-reactive and sulfhydryl-reactive crosslinkers (such as, forexample, SPDP, LC-SPDP, and Sulfo-LC-SPDP; SMPT and Sulfo-LC-SMPT; SMCCand Sulfo-SMCC; MBS and Sulfo-MBS; SIAB and Sulfo-SIAB; SMPB andSulfo-SMPB; GMBS and Sulfo-GMBS; SIAX and SIAXX; SIAC and SIACX; NPIA),carbonyl-reactive and sulfhydryl-reactive crosslinkers (such as, forexample, MPBH, M 2 C 2 H, PDPH), amine-reactive and photoreactivecrosslinkers (such as, for example, NHS-ASA, sulfo-NHS-ASA, andsulfo-NHS-LC-ASA; SASD; HSAB and Sulfo-HSAB; SANPAH and Sulfo-SANPAH;ANB-NOS; SAND; SADP and Sulfo-SADP; Sulfo-SAPB; SAED; Sulfo-SAMCA;p-Nitrophenyl Diazopyruvate; PNP-DTP), sulfhydryl-reactive andphotoreactive crosslinkers (such as, for example, ASIB, APDP,Benzophenone-4-iodoacetamide, Benzophenone-4-maleimide),carbonyl-Reactive and Photoreactive Crosslinkers (such as, for example,ABH), carboxylate-reactive and photoreactive crosslinkers (such as, forexample, ASBA), arginine-reactive and photoreactive crosslinkers (suchas, for example, APG)), or trifunctional crosslinkers(4-Azido-2-nitrophenylbiocytin-4-nitrophenyl ester, Sulfo-SBED,MTS-ATF-Biotin and MTS-ATF-LC-Biotin, Hydroxymethyl PhosphineDerivatives).

In some specific applications, it is sufficient (and sometimes evennecessary) to provide identical polymeric material for making thenanoparticles. However, in other applications, it is preferably to havedifferent polymeric material in the nanoparticle. In one specificexample, it is contemplated that two different polymeric material beprovided, each one only differing in the type of peptide that they bear.For example, the two different polymeric material can each bear either atargeting/penetrating peptide that can bind to a cell surface orcell-associated receptor (such as MGF, CP-15 or P160 for example) or atargeting/penetrating peptide that can facilitate the translocation ofthe non-viral particle across a cellular membrane (such as TAT forexample).

Use of the Non-Viral Nanoparticles

The non-viral nanoparticles described herein can be used to specificallydeliver an anionic agent to a cell. In order to do so, an appropriateamount of the non-viral nanoparticle is contacted with the cell andincubated in conditions appropriated to allow the entry of thenanoparticles into the cell. In some embodiments, such contact betweenthe nanoparticles and the cell occurs in vitro.

In other embodiments, the cell remains in vitro after the contact (andfollowing the entry of the nanoparticles). However, in some embodiments,the contacted cell can be introduced into an individual in need thereof.Such embodiments may be useful for providing ex vivo gene therapy to theindividual. For example, cells explanted from an individual patient(e.g., lymphocytes, bone marrow aspirates, and tissue biopsy) oruniversal donor hematopoietic stem cells, followed by re-implantation ofthe cells into the individual, usually after selection for cells whichhave incorporated the nanoparticles. In one embodiment, stem cells areused in ex vivo procedures for cell transfection and gene therapy. Theadvantage to using stem cells is that they can be differentiated intoother cell types in vitro, or can be introduced into a mammal (such asthe donor of the cells) where they will engraft at an appropriatelocation (such as in the bone marrow).

In alternative embodiments, the non-viral nanoparticle is formulated foradministration prior to being administered to the individual. Suchformulation can provide a systemic administration (intravenous,intraperitoneal, intramuscular, subdermal, or intracranial infusion) ora topical administration (cutaneous, intranasal, mucosal, etc.).Although more than one route can be used to administer thenanoparticles, a particular route can often provide a more immediate andmore effective reaction than another route. The nanoparticles of thepresent invention may be administered, either orally or parenterally,systemically or locally. For example, intravenous injection such as dripinfusion, intramuscular injection, intraperitoneal injection,subcutaneous injection, suppositories, intestinal lavage, oral entericcoated tablets, and the like can be selected, and the method ofadministration may be chosen, as appropriate, depending on the age andthe conditions of the patient. Since some of the nanoparticles aredesigned to cross efficiently biological barrier (such as the BBB, themucosa or/and the skin), the nanoparticles can be formulated forcontacting such biological barrier.

In some embodiments, the nanoparticles are used for delivering atherapeutically effective amount of the anionic agent to the brain of anindividual in need thereof. The nanoparticles can be formulated as nasaldrops for intranasal administration. Such embodiments includes theprevention, treatment and/or alleviation of symptoms associated to aneurodegenerative disease or a brain cancer. Exemplary embodiments ofthe neurodegenerative diseases include but are not limited tospinocerebellar ataxia, Huntington's disease, Parkinson's disease,Alzheimer's disease, dementia and/or amyotrophic lateral sclerosis. Alsocontemplated herein are non-viral nanoparticles for the delivery ofagents to the brain that can also be used for the prevention, treatmentand/or alleviation of symptoms associated to a neurodegenerative diseaseor a brain cancer

In other embodiments, the nanoparticles can be used for delivering atherapeutically effective amount of an anionic agent to a cancer cell(such as a carcinoma cell). The nanoparticles can be formulated forintravenous/intraperitoneal/intratumor administration prior to beingadministered to the individual. The nanoparticles can be used for theprevention, treatment and/or alleviations of symptoms associated with aproliferative disease, such as cancer (e.g., a colon cancer).

Also contemplated herein are non-viral nanoparticles for the delivery ofagents to the brain that can also be used for the prevention, treatmentand/or alleviation of symptoms associated to a cancer. The nanoparticlescan be administered alone or in conjunction with other anti-neoplasticcompounds. The use of the nanoparticles can also be combined withradiation therapy.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

Example I—Production and Characterization of the Nanoparticles

Material and methods. Low Molecular weight (LMW) chitosan (M.W. 10 KDa)was obtained from Wako (Richmond, Va., USA), having a viscosity of 5˜20cP (at room temperature) and degree of deacetylation of 80.0%.Polyethylene glycol monomethyl ether (mPEG): medium molecular weight (2000 Da), phthalic anhydride, pyridine, toluene, hydrazine monohydrate,succinic anhydride, ethanethiol, aluminium chloride, sodiumtripolyphosphate (TPP), agarose, ethidium bromide (10 mg/ml) and glacialacetic acid of analytical grade were obtained from Sigma (Oakville, ON,Canada). Anhydrous N,N-Dimethylformamide (DMF),1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (EDC),4-(Dimethylamino) pyridine (DMAP), sodium hydroxide (NaOH), methanol,diethyl ether, chloroform and concentrated hydrochloric acid wereobtained from Thermo Fisher Scientific (Ottawa, ON, Canada). Thionylchloride (SOCl₂) was obtained from VWR (Mississauga, ON, Canada). Thepeptides, TAT peptide (NH₂-RKKRRQRRR-NH₂) (SEQ ID NO: 4) M.W. 1339.40,and MGF peptide (NH₂-YQPPSTNKNTKSQRRKGSTFEEHK-NH₂) (SEQ ID NO: 1) M.W.2848.14, were synthesized by Sheldon Biotech, McGill University. MouseNeuro2a cells and EMEM media were obtained from Cedarlane laboratories(Burlington, Ontario, Canada). MTS cytotoxicity assay kit was obtainedfrom Promega (Madison, Wis., USA). Subcloning DH5-alpha competent cellsand Lipofectamine 2000, TrackIt 10-bp DNA ladder (0.5 μg/μl), Pre-castNupage 4-12% Bis-Tris gels, Nupage MES running buffer, Nupage transferbuffer, Nupage LDS sample buffer (4×), nitrocellulose membrane 0.45 μm,blotting filter paper and magic mark (1 Kb) protein ladder were obtainedfrom Invitrogen (Burlington, ON, Canada). Orange dye solution (6×) wasobtained from Fermentas (Burlington, ON, Canada). pEGFP^(ataxin1)(E3-82Q) plasmid was obtained from Dr. Zoghbi Huda from Baylor Collegeof Medicine, Houston, USA. Plasmid purification Maxi kit is from Qiagen(Mississauga, Ontario, Canada). Primary antibodies: Ataxin-1 (L-19) goatpolyclonal antibody, Actin (1-19) goat polyclonal antibody and(HRP)-conjugated donkey anti-goat IgG antibody were procured fromSantacruz Biotechnology (Santa Cruz, Calif., USA). Ataxin-1 siRNA (h)was procured from Santacruz Biotechnology (Santa Cruz, Calif., USA).SiGLO red (tagged with Cy3), was obtained from Thermo Fisher Scientific(Ottawa, ON, Canada). Scrambled/Non targeting (NT) siRNA tagged withbiotin was obtained from Dharmacon (Lafayette, Colo., USA).

Commercially available low molecular weight chitosan (CS) with anaverage molecular weight of 10 000 Da and with degree of deacetylation(DA) of 80% (1.00 g) was added to a solution of phthalic anhydride (PH)(2.76 g) in 20 ml of N,N-Dimethylformamide (DMF) in order to protect theamine groups of chitosan. The mixture was stirred for 8 hours at 110° C.under nitrogen atmosphere (Kurita et al., 2002). The resultant product(c) in FIG. 1 was cooled to room temperature and precipitated in icecold water. The precipitate was filtered, washed with methanol overnightand vacuum dried. This is an important step in carrying forward anyfurther chemical reactions with chitosan, as chitosan being soluble onlyin acidic conditions becomes soluble in most organic solvents such asDMSO, DMF, THF, pyridine etc. after protection with phthalic anhydride.

Phthaloylation of the chitosan polymer. ¹³C NMR data confirms successfulN-phthaloylation of the product. Product (c) in FIG. 1 shows theschematic representation of the N-phthaloyl chitosan. The TotalSuppression of Side bands (TOSS) mode exhibit peaks at 124.62 ppm,131.70 ppm, 134.42 ppm corresponding to phenylene, and 169.66 ppm andcarbonyl group of phthaloyl group and the TOSS-dipolar dephasing (TOSDL)mode confirms the absence of CH and CH₂ peaks due to their shortrelaxation time and presence of only two peaks 131.63 ppm and 169.89 ppmassigned to C 1, 2 and carbonyl of phthaloyl group respectively. The IRdata shows distinct sharp peaks at 1774 cm⁻¹ and 1702 cm⁻¹ correspondingto imide of phthaloyl group in FIG. 2 . The phthaloyl chitosan preparedby this method becomes gel-like when precipitated in water, whichsupports the data shown by Kurita, of formation of a uniform structureof phthaloylated chitosan (Kurita et al., 2002).

Pegylation of chitosan: Synthesis of mPEG-COCl. To 10% solution ofmonomethyl ether PEG (OH-PEG-OCH₃) (M.W. 2 000) in toluene was convertedto carboxylate-terminated PEG (COOH-PEG-OCH₃) by reacting it with fourmolar excess of succinic anhydride (dissolved in pyridine). The abovereaction was set up under anhydrous conditions and refluxed for 6 hours.The product was precipitated with ether, filtered and againre-precipitated twice with chloroform and diethyl ether and finallyvacuum dried. The final product obtained (a), is shown in FIG. 1 .

FIG. 3 represents the IR spectra that shows the presence of carboxylicpeak at 1732 cm⁻¹, confirming the presence of (—C═O) carbonyl groups onPEG. To mPEG-COOH obtained in the previous reaction, 2 fold molar excessof SOCl₂ was added under N₂ atmosphere to form PEG acyl chloride(COCl-PEG-OCH₃) (product b in FIG. 1 ). The reaction was refluxed toboil for 6 hours, followed by degassing to remove excess SO₂ and thionylchloride.

Pegylation of chitosan: Synthesis of CSPH-O-mPEG (PEGylating phthaloylchitosan). This product (b) PEG acyl chloride (COCl-PEG-OCH₃) obtainedin previous reaction is used as an active intermediate to be furtherconjugated to hydroxyls of 2-N-phthaloyl chitosan (Product c). For thisreaction, phthaloyl chitosan (CSPH) (300 mg), product (c), was soaked inpyridine solution overnight and added to mPEG acylchloride (10 g),product (b), in toluene. The reaction was stirred for 2 hours at roomtemperature and then refluxed to boil for 24 hours (Lin et al., 2007).The resultant product was allowed to cool at room temperature,precipitated in methanol and vacuum dried to yield product (d) in FIG. 1.

The FTIR spectra in FIG. 4A shows PEG grafted phthaloylated chitosanwith characteristic peaks at 2873 cm⁻¹ (C—H stretching), 1068 cm⁻¹ (C—Ostretching), 1491 cm⁻¹, 1451 cm⁻¹ and 1254 cm⁻¹ belong to PEG. Also thereduction in hydroxyl peaks of chitosan at 3200-3500 cm⁻¹, indicates thegrafting of PEG onto chitosan forming 2-N-phthaloyl chitosan-O-mPEG,product (d) in FIG. 1 .

Tagging a peptide to a pegylated chitosan polymer. To conjugate peptideonto PEGylated phthaloyl chitosan, the methoxy group of PEG wasconverted to hydroxyl group by following the procedure as mentioned inLin et al. (2007). In brief equi-molar ratios of CS-mPEG and aluminiumchloride were reacted together for 12 hours at room temperature in 20 mlof ethanethiol. The reaction mix was diluted with water, acidified with10% HCl, filtered and extracted thrice with dichloromethane to yieldproduct (e) in FIG. 1 .

The hydroxyl groups formed (CSPH-PEG-OH) were further converted tocarboxyl groups by reacting it with 4 molar excess of succinic anhydridein toluene at 100° C. for 12 hours. The product obtained as (f) in FIG.1 was cooled at room temperature, precipitated with methanol, filteredand vacuum dried. The carboxylic terminal groups of this product (f)i.e. CS-PEG-COOH (12 mM) was conjugated to the equimolar ratio ofpeptide-NH₂ (12 mM) in the presence of EDC (15.5 mM) and DMAP (1.2 mM)in 1 ml of DMF for 24 hrs. This reaction mix was dialyzed againstdeionized water for 2 days, using a dialysis tube with molecular weightcut-off of 3 500 Da. The resultant product (g) was obtained as shown inFIG. 1 .

The IR spectra in FIG. 4B shows the appearance of a new peak at 1659cm⁻¹ that shows the presence of amide bonds and confirms the conjugationof peptide onto the polymer forming 2-N-phthaloylchitosan-O-PEG-CONH-peptide, product (g) in FIG. 4 .

Deprotection of chitosan polymer-CS-PEG-peptide (TAT/MGF). As a laststep of the synthesis, the amine groups of chitosan were deprotectedusing 5% hydrazine monohydrate in DMF. The reaction was carried out at100° C. for an hour under inert conditions. The mixture was allowed tocool at room temperature and was dialyzed against deionized water for 2days, using a dialysis tube with molecular weight cut-off of 3 500 Da.The sample obtained after dialysis was vacuum dried and marked asproduct (h) in FIG. 1 . Hydrazine monohydrate being basic in naturecauses destabilization of the phthaloyl moiety by creating an excessalkaline condition of pH>12.

The removal of phthaloyl group from the polymer was confirmed by FTIR,where the absence of peaks at 1775 cm⁻¹ and 1710 cm⁻¹ confirmed thecomplete dissociation of phthalimido group from chitosan (FIG. 4C). Itwas observed that the reaction conditions followed in the scheme atcertain steps had no deleterious effects on any of the previous bondsformed. The peak representing the presence of amides due to the peptideshifts from 1659 cm⁻¹ to 1644 cm⁻¹ and the peak at 1582 cm¹ (amide II)belongs to the chitosan. The peaks at 2918 cm⁻¹ and 1061 cm⁻¹ refers tothe presence of (CH₂) groups and (C—O stretch) of PEG respectively asobserved in FIG. 4 c . The final product was dialyzed for 2 days againstdeionized water in order to get rid of any impurity or byproduct leftduring the reaction.

Polymer characterization. The polymer CS-PEG-peptide was characterizedand analyzed at every step of its derivation through FTIR (Perkin ElmerSpectrum BX), ¹³C (Varian 300 MHz broadband NMR) and ¹H (Mercury 400 and500 MHz NMR) and Transmission Electron Microscopy (Philips EM410 TEM).

FIG. 5 represents the ¹H NMR spectra of 2-N-phthaloyl chitosan (FIG.5A), 2-N-phthaloyl chitosan-O-mPEG (FIG. 5B), 2-N-phthaloylchitosan-O-PEG-CONH-TAT (FIG. 5C) and chitosan-O-PEG-CONH-TAT (FIG. 5D).The chemical shift at δ 7.78 belongs to the aromatic protons of thephthaloyl moiety, which is present in the spectra of 2-N-Phthaloylchitosan-O-mPEG, 2-N-phthaloyl chitosan-O-PEG-CONH-TAT but disappearedin chitosan-O-PEG-CONH-TAT spectrum. The multiple peaks of oxymethylgroups in PEG at δ 3.3 to 3.7 cover over the signals of pyranose ring ofchitosan in all the three spectras. The weak and broad peak at δ 4.3-4.5were from the protons of —NH—CH(CH₂)—CO— in TAT peptide as observed inspectrum 2-N-phthaloyl chitosan-O-PEG-CONH-TAT. The peak at δ 2.7-2.8came from the protons of —CH₂—NH—NH—NH₂ in arginine and the weak andmultiple peaks at δ 1.3-1.7 came from the —CH₂—CH₂—CH₂—NH—NH—NH₂ inarginine as observed in spectra 2-N-phthaloyl chitosan-O-PEG-CONH-TATand chitosan-O-PEG-CONH-TAT. The multiple peaks at δ 7.0-8.0 belong tothe amines in TAT peptide sequence (FIG. 5E).

Preparation of siRNA complexes with CS-PEG-PEPTIDE (TAT/MGF)nanoparticles. FIG. 6 represents TEM images of the polymerchitosan-O-PEG-CONH-peptide dissolved in dilute acetic acid at pH 6 at aconcentration of 0.5 mg/ml, which was sonicated for 10 minutes beforebeing observed under the transmission electron microscope (TEM). FIG. 6Ashows the TEM image of the polymer PEGylated chitosan polymer atmagnification 53 800× and FIG. 6B is the peptide tagged PEGylatedchitosan polymer at magnification 53 800×. The spherical shape of theparticles formed is attributed to the conjugation of PEG on chitosan.FIG. 6C shows effect of acidic pH (5.5) on peptide tagged PEGylatedchitosan polymer at magnification 70 700× formed before complexingsiRNA. The polymer appeared to disperse and disintegrate at acidic pH.

To form siRNA complexed nanoparticles, scrambled/non-targeting (NT)siRNA (siGLO-red) was mixed with TPP at pH 3, as described previously(Malhotra et al., 2009) and then added drop-wise to the polymer solutionof chitosan-PEG-peptide at a concentration 0.5 mg/ml. Finalconcentration of siRNA used was 2 μg/ml. The solution was stirred for anhour at room temperature. The nanoparticles obtained were viewed underTEM.

FIG. 6D shows nanoparticles formed with the modifiedChitosan-PEG-peptide polymer, without siRNA, using TPP as a crosslinker.The nanoparticles range in the size of 50-100 nm. FIGS. 6E and 6Frepresent nanoparticles formed with unmodified chitosan complexing siRNAand Chitosan-PEG-peptide polymer complexing siRNA respectively. Onencapsulating siRNA, the nanoparticles obtained fromchitosan-PEG-peptide polymer were smaller than 20 nm and appeared morespherical and monodispersed, (FIG. 6F) as compared to the particlesobtained by unmodified chitosan polymer which were 50-80 nm andnon-spherical (FIG. 6E). The reduction in size is because of theincrease in amine groups due to the peptides, which interacts moreefficiently with negatively charged siRNA and thus the amount of siRNAcomplexed increases as well. The increased encapsulation efficiencyyields smaller particles.

Example II—In Vitro Transfection with the Nanoparticles

Transfection efficiency of the nanoparticles complexing siGLO red(Cy3-labelled transfection indicator) was performed on mouseneuroblastoma cells (Neuro 2a). 100% transfection efficiency wasachieved with chitosan-PEG-TAT nanoparticles encapsulating siGLO-red inmouse neuroblastoma cells seeded at a density of 20 000 cells per wellin a 96 well plate, supplemented with Eagles minimum essential medium(EMEM) with 10% FBS (FIG. 7 ). The fluorescence was achieved after 4hours of transfection and was observed under fluorescence microscope(Nikon Eclipse TE2000-U) at a wavelength of 660 nm.

The cytotoxicity assay was performed using MTS assay on cells seeded in96 well plate after 4 hours of transfection with different treatmentgroups (FIG. 8 ). The results indicate minimal toxicity with chitosannanoparticles modified with PEG alone and with chitosan nanoparticlesmodified with PEG and peptide both as compared to the unmodifiedchitosan nanoparticles. Without wishing to be bound to theory, it isbelieved that the presence of PEG and peptide which are hydrophilic innature and pose no adverse or toxic effect on the cells. It is alsobelieved that PEG reduces the steric-hindrance and TAT is an excellentcell-penetrating peptide. It is also noted that chitosan polymer itselfwas observed to be toxic to the cells, whereas anionic agent siGLO andthe cross linker TPP showed no/minimal adverse effects on the cells.

Plasmid purification: Ataxin-1 cDNA. The plasmid DNA containing ataxin-1cDNA, purified and resuspended in Tris-EDTA buffered solution[pGFP-ataxin (Q82)] was a kind gift from Dr. Zoghbi Huda, which was usedfor the transformation experiment in DH5-alpha competent cells by meansof chemical transformation also known as heat shock transformationmethod. The procedure for chemical transformation was followed accordingto the manufacturer's protocol (Invitrogen). The colony selectiveantibiotic for pGFP-ataxin was kanamycin (30 μg/ml). Stock cultures oftransformed plasmid in DH5-alpha cells were made in sterilized 80%glycerol and were stored at −80° C. Plasmid purification was performedfollowing the protocol of Qiagen's maxi kit. The purified plasmidobtained was quantified for its concentration and purity. The percentagepurity of the plasmid was quantified using UV spectrophotometer. Theratio of Abs 260/280 obtained was 1.8 which signifies that the plasmidwas purified and the concentration obtained was 266 μg/ml forpEGFP.ataxin1.

Overexpression/Suppression of ataxic proteins in Neuro2A cells.Transfection studies were performed using a functional siRNA againstataxin gene. 100 000 cells were seeded in a 12-well plate, supplementedwith EMEM containing 10% FBS. The cells were incubated for 24 hours at37° C., 5% CO₂. After 24 hours, the media was replaced with FBS freeEMEM medium and the cells were transfected with pEGFP-ataxin1 at aconcentration of 250 ng/μl using Lipofectamine 2000™ (Invitrogen)according to manufacturer's protocol. Optimal transfection efficiencywith lipofectamine was determined experimentally. After 6 hours oftransfection, the media was replaced with complete growth EMEM mediumcontaining 10% FBS. Ataxin1-siRNA (Santacruz Biotechnology) wascomplexed with chitosan-PEG-peptide nanoparticles as previouslydescribed. The Ataxin1-siRNA transfection with nanoparticles wasperformed on cells over-expressing ataxin protein. After 24 hours ofoverexpression, 40 μM of Ataxin1-siRNA complexed with nanoparticles wasused to transfect neuronal cells per well (12 well plate) containing FBSfree EMEM medium. Further to nanoparticles transfection after 6 hours,the media was again replaced with complete growth EMEM medium containing10% FBS. Empty nanoparticles and nanoparticles containing scrambledsiRNA (siGLO) were used as positive controls. The cells were harvestedfor ataxin protein estimation after 24 and 48 hours.

Protein extraction and Western Blot. To estimate the amount of silencingby functional siRNA delivered through Chitosan-PEG-peptidenanoparticles, cultured cells were washed twice with cold PBS and lysedusing 2× Laemmli Buffer (4% SDS, 20% glycerol, 10% 2-β-mercaptoethanol,0.004% bromophenol blue, 0.125 M Tris HCl, pH 6.8) at 4° C. on anorbital shaker. The cells were scrapped after 20 minutes (on ice). Theextracted protein samples were heated at 70 to 100° C. for 10 minutes,before loading on gels. Procedure of western blot was performedaccording to the manufacturer's protocol (Invitrogen). Pre-cast NupageBis-Tris (4-12%) gels were used for running the protein samples. Magicmark 1 Kb protein ladder was used as a standard. The gel waselectrophoretically transferred to nitrocellulose membrane using SemiDryblot apparatus (Invitrogen).

The membrane was incubated for 1 hr in 5% non-fat powdered milk in 0.2%TBST. The membrane was then incubated overnight with goat ataxin-1 IgGpolyclonal antibody (1:5 000 dilution; Santacruz Biotechnology). Afterthree washes in TBST, the membrane was incubated with horseradishperoxidase (HRP)-conjugated donkey anti-goat IgG antibody (1:2 000dilution; Santacruz Biotechnology). The membranes were then washed threetimes in TBST followed by detection of signal with a chemiluminescencedetection kit (Roche) (FIG. 9 ).

To control the protein loading, the membrane was reprobed with antibodya-Actin (1:10 000; Santa Cruz Biotechnology). As observed in FIG. 9 ,samples were run after 24 and 48 hours of transfection. Sample A and Cwere positive controls with empty and scrambled siRNA (siGLO) containingCS-PEG-TAT nanoparticles respectively and sample B had Ataxin1-siRNA.The silencing was observed after 48 hours in sample B. These resultsindicate that nanoparticles prepared using the proposed synthetic schemesuccessfully and efficiently delivered siRNA, leading to gene silencingagainst ataxin 1 in neuronal cells in vitro.

Example III—In Vivo Use of the Nanoparticles for Brain Delivery

Four weeks old C57BL/6J male mice weighing 10 to 15 g were purchasedfrom Jackson laboratories (Bar Harbor, Me., USA) and housed in anenvironment with controlled temperature (22° C.), humidity, and a 12 hlight/dark cycle at McGill's Animal care facility. The animal experimentwas conducted as per the protocol approved by the Animal care committeeat McGill University (Montreal, QC, Canada). Standard mouse chow pelletsand water were supplied ad libitum. Animals were acclimatized for a weekbefore the start of the experiment.

Animal Study, tissue processing and staining. Animals were randomizedinto 4 groups to receive treatment formulation consisting of biotintagged scrambled siRNA, complexed with CS-PEG-peptide (TAT/MGF)nanoparticles (two animals/group). One animal in each group received PBSand was treated as control. CS-PEG-peptide (TAT/MGF) nanoparticleformulations complexing biotin-siRNA were concentrated to 4 differentdoses at: (a) 0.25 mg/kg, (b) 0.5 mg/kg, (c) 1 mg/kg and (d) 2 mg/kgsiRNA using Am icon Ultra-15 centrifugal filters (MW cut-off 3 000Daltons, Millipore) prior to dosing. The animals were anesthetized with75-100 μl of cocktail comprising ketamine (100 mg/kg), xylazine (10mg/kg) and acepromazine (3 mg/kg) via intraperitoneal administration. Atotal of 30 μl of the nanoparticle formulation was administeredintranasally (2 μl/drop) over 15-20 minutes period, once a day. Theexperimental end points were 4, 16, 24 and 48 hrs. Before each end pointthe animals were anesthetized using the above mentioned anestheticcocktail and perfusion fixed with 4% paraformaldehyde (PFA) (SigmaAldrich, Canada). Brain, Lungs, Heart, Stomach, Kidney and Liver wereharvested and kept at 4° C. in 4% PFA for 48 hrs. After which thetissues were trimmed to 3 mm thick sections and stored at 70% ethanol inhistology cassettes. The tissues were paraffin embedded and processedinto 4 μm thick section on slides at the histology core facility (TheRosalind and Morris Goodman Cancer Research Centre, McGill University).The tissue section on slides were stained with Vectastain elite ABC kit(Vector laboratories; Burlingame, Calif., USA) as per the manufacturer'sprotocol and Diaminobenzidine (DAB) was used as a substrate to assessthe presence of biotin (brown staining). Hematoxylin was used as acounterstain and slides were mounted with permount (Vector laboratories;Burlingame, Calif., USA) and observed under compound microscope (LeicaDM500; Ontario, Canada) at ×400 magnification.

FIG. 10 (I) represents histopathological sections of brain tissue(cerebral cortex) at 4 hrs time point with different doses, containing(A) 0.25 mg/kg, (B) 0.5 mg/kg, (C) 1 mg/kg, (D) 2 mg/kg and (E) PBS(control). The dark brown stained pyramidal neuronal cells obtained with0.5 mg/kg of scrambled biotin-siRNA complexed nanoparticles, ensured thedelivery of siRNA in the neuronal cells of cerebral cortex (p=0.0001)and in the Purkinje cells of the cerebellum (p=0.0001) as compared tothe untreated control. Other animals that received 0.25 mg/kg ofscrambled biotin-siRNA showed faint staining in the neuronal cells ofcerebral cortex (p=0.006), this is justified by the fact that dose A(0.25 mg/kg) was half the concentration of dose B (0.5 mg/kg). Whereas,animals that received 1 and 2 mg/kg of scrambled biotin-siRNA dose, didnot show any staining in the tissue. The reason was interpreted, aswhile concentrating the nanoparticle formulation from a range of 10-20ml solution to 30 μl before dosing, the process of concentrating led toclumping and aggregation of the nanoparticles, which resulted inincrease in nanoparticles size and it could not penetrate the neurons.FIG. 10 (II) represents the quantitative analysis of the tissues usingImageJ, which calculates the mean percentage area of the dark brownstained cells using image J software. Thus, this study determined that,under these experimental conditions, the optimal dose for the developednanoparticles formulation administered intranasally was dose B at 0.5mg/kg. In addition, these results indicated that the nanoparticleformulation can be used for the successful delivery of the biotin-siRNAwith high efficiency and high target selectivity.

FIG. 11 (I) represents histopathological sections of brain tissue(cerebral cortex) of mouse having received dose B (as described in FIG.10 ) at 0.5 mg/kg at different time points, (A) 4 hrs, (B) 16 hrs, (C)24 hrs and (D) 48 hrs. As observed in the figure, the staining with dose0.5 mg/kg shows significant dark brown staining in the pyramidal neuronsof the cerebral cortex and Purkinje cells of the cerebellum (p=0.0001)at 4 hrs (A). The staining was observed only until 16 h in the cerebralcortex (p=0.0001) and faded thereof, with no staining observed at 24 and48 h. The result was quantified using Image J as represented in FIG. 11(II). The time dependent study was conducted to determine and monitorthe expression of siRNA after being delivered by nanoparticleformulation via intranasal route of administration. These resultsindicate that, in these experimental conditions, the nanoparticleformulation containing 0.5 mg/kg siRNA was successfully delivered toneuronal cells within 4 hours. These results also indicate that theintranasally administered nanoparticle formulation is, under theseexperimental conditions, cleared after 16 hrs.

FIG. 12A represents histopathological sections of different organs withtreatment dose B (as described in FIG. 10 ) comprising nanoparticleformulation at siRNA dose 0.5 mg/kg (left column) and control group withPBS (right column) after 4 hours. As observed in the figure, thestaining in the brain tissue was highly significant with 0.5 mg/kgscrambled biotin-siRNA/nanoparticle dose in both cerebral cortex andcerebellum (p=0.0001) and also with 0.25 mg/kg but only in the cerebralcortex (p=0.006), as represented in FIG. 4B. The staining withbiotin-siRNA/nanoparticle dose at 0.5 mg/kg was also observed to targetheart sarcomeres (p<0.01), with significance as compared to other doseconcentrations; 0.25 mg/kg (p=0.403), 1 mg/kg (p=0.562), 2 mg/kg(p=0.999) (FIG. 12 B). Renal cells in the medulla region of the kidneyand hepatic cells also showed brown-colored staining in the cells, with0.5 mg/kg of scrambled biotin-siRNA/nanoparticle formulation (p=0.0001)as compared to the untreated control (FIG. 4B). The glandular cells ofthe stomach and alveoli in lungs showed no significant difference ascompared with the untreated control. Among all the concentrations ofdifferent treatment doses tested, the highest staining was observed with0.5 mg/kg of scrambled biotin-siRNA/nanoparticle formulation in thecerebral cortex and cerebellum (p<0.01), when compared with staining inother organs, except the heart. FIG. 13 represents the enlarged view ofcerebral cortex and purkinje cells. However, in the cerebral cortex, theanterior olfactory bulb, hippocampus, thalamus, hypothalamus were alsofound to be well stained.

As per the nanoparticle formulation of CS-PEG-TAT/MGF, the MGF peptidewas used as targeting ligand for purkinje cells. The MGF peptide is asplice variant of IGF and has been shown to have affinity towardsneuronal cells and myocardial tissues. Thus the staining in the heartcould be the due to the affinity of MGF peptide targeting the heartsarcomeres. Following the intranasal route of administration, a fairamount of nanoparticles was expected to target lungs through the airwaypathway. Renal, hepatic cells have been shown to have stained ascompared to the control sections. It is to be noted that no staining wasobserved in any tissue sections at 24 hrs and 48 hrs time point. Thiscould be due to the degradation of the biotin—siRNA molecule by thattime and at 16 h, except brain tissues (cerebral cortex) no other tissuesection was observed to have staining in the cells.

Cytotoxicity—TUNEL assay on animal tissues. Analysis of apoptotic cellsin tissues was performed using Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) staining (Promega,Madison, Wis., USA) as per the manufacturer's protocol after apretreatment procedure performed to block biotin-siRNA. In brief, thetissue sections in paraffin block were dewaxed in xylene and rehydratedin decreasing concentrations of ethanol and washed with PBS. The tissuesections were then incubated with Streptavidin-HRP reagent (RocheDiagnostics) for 10 minutes, washed in PBS. After which the sectionswere incubated with 3% H₂O₂ for another 10 minutes and again washed inPBS. This step was included to block biotin-siRNA already present in theneuronal cells and to avoid any false positive results. After this step,the protocol of TUNEL assay was performed as per the manufacturer'sinstructions. The tissue sections were counterstained with hematoxylin,washed in distilled water, dehydrated and mounted with permount (Vectorlaboratories; Burlingame, Calif., USA) to be observed under compoundmicroscope (Leica DM500; Ontario, Canada) at ×400 magnification.

FIG. 14 represents histopathological sections of different organ tissuesfrom animal receiving nanoparticle formulation containing biotin taggedscrambled siRNA as a dose at 0.5 mg/kg of animal weight, euthanizedafter 4 hrs. The tissues did not show any apparent staining in thecells, which confirms no toxicity/apoptosis of cells due to theadministration or presence of the nanoparticle formulation.

Without wishing to be bound to theory, FIG. 15 represents a potentialmechanism of cellular uptake of surface functionalized, nanoparticlethrough a targeted receptor-mediated endocytosis pathway, withendocytotic release from the endosome due to “proton sponge” effect.Following the endosomal disruption, the delivered therapeutic moleculegets released intra-cellularly.

Example IV—In Vivo Use of the Nanoparticles for the Treatment ofAlzheimer's Disease

Neurodegenerative diseases are characterized by progressive, age-relatedloss of specific subsets of neural cells, which lead to diverse clinicalphenotypes depending on the underlying anatomical involvement. Theetiology of neurodegenerative diseases is most often multifactorial,likely a result of gene—environmental interaction, which may leaddiseases like Parkinson's, Alzheimer's, Huntington's, Amyotrophiclateral sclerosis (ALS) and Spinocerebellar Ataxia (SCA). These diseasestend to progress slowly over the time and generally target olderpopulation. Alzheimer's is the most common form of dementia, which isincurable and degenerative. It is predicted to affect 1 in 85 peopleglobally by 2050. As per the 2008 clinical trial report, it is uncertainif any of the intervention strategies tested among 500 clinical trials,are likely to show promise for the identification and treatment ofAlzheimer's disease (AD). The mean life expectancy of an individual isdrastically reduced to 7 years after the onset of the disease.Currently, there is no cure available that can halt the progression ofthe disease. However, therapies and drugs are available in the marketsthat merely mitigate the symptoms of the disease.

Alzheimer's disease is characterized by the presence of misfoldedprotein in the form of senile plaques and neurofibrillary tangles in thebrain. The senile plaques are composed of dense, insoluble deposits ofamyloid-beta peptide in and around the neuron. These peptides are thefragment of a larger transmembrane protein called amyloid precursorprotein (APP). APP is essential for neurons growth survival and postinjury repair. The formation of amyloid-beta peptides is initiated by asequential cleavage of APP by an enzyme, protease beta-secretase, alsoknown as BACE1 (beta site APP cleaving enzyme) and then by agamma-secretase, an aspartyl protease complex, which generates toxicC-terminal fragments inside the cell and releases a fragment calledamyloid-beta peptide extra cellularly. Neurofibrillary tangles areaggregates of the microtubule-associated tau protein which has becomehyperphosphorylated and accumulate inside the cells. The underlyingpathological mechanism of AD is still unknown but the accumulation ofamyloid-beta peptides is thought to be the central triggering event ofthe disease, which is believed to disrupt the cell's calcium ionhomeostatis, leading to apoptosis. Amyloid-beta is also known toaccumulate in the mitochondria of affected cells and inhibit certainenzyme functions that further inhibit the utilization of glucose byneurons. Moreover, alteration in the distribution and expression ofbrain derived neurotrophic factors (BDNF) has also been observed to beassociated with AD.

The identification of these biological and pathological abnormalitiestriggered the studies on recognizing the genes responsible for causinginherited forms of AD. On such form is autosomal dominant familialAlzheimer's disease, which is caused due to the mutations in the APP andcomponents of gamma-secretase (Presenilins 1 and 2) that lead toincreased production of amyloid-beta 2 protein, the main component ofsenile plaques. Several transgenic animal models have been developedbased on various genetic mutations to understand the aetiology andpossible pathological mechanism of the disease and to investigatevarious therapeutic options.

Advancement in RNAi therapy has facilitated the understanding ofpathobiological mechanisms of the disease with most of the researchesfocusing on phenotype rescue due to dominantly acting mutant genes andloss-of function analysis. Based on the aetiology of the AD, the keytargets for RNAi therapy are assumed to be APP, BACE and gamma-secretaseand tau, which can eliminate the production of toxic C-terminalfragments and amyloid-beta peptides. siRNAs has been delivered to thecentral nervous system both naked or with the help of some transfectionreagents in-vivo, targeting different molecular targets in differentparts of the nervous system, showing effective gene silencing. Directdoses of siRNAs administered intrathecally or intracerebro-ventricularlypose a widespread inhibition of molecular targets that are broadlyexpressed in different parts of the brain but may also lead tooff-targeting. A significant limitation is the inability of thetherapeutic molecules to cross the blood-brain barrier and otherphysiological barriers. Thus the application of a novel therapeuticmodality also depends on the development of an efficient and clinicallyfeasible means of administration. Though, various siRNA deliverystrategies have been explored, but they have not proved to be aseffective and have created concerns with safety issues, thus a polymericapproach of delivering siRNA molecules, which is target specific,multifunctional, biodegradable and biocompatible is more appealing. Wehave developed a self-assembled, functionalized receptor targetingnanoparticles from chitosan that are biodegradable and biocompatible innature and perform target specific delivery of siRNA, when deliveredintranasally to the cerebral cortex of the brain.

Materials. Chitosan (M.W. 7 to 10 KDa) with a viscosity of 5 to 20 cP(at room temperature) and a degree of deacetylation of 80.0% waspurchased from Wako (Richmond, Va., USA). Polyethylene glycol monomethylether (mPEG): medium molecular weight (2 000 Da), phthalic anhydride,pyridine, toluene, hydrazine monohydrate, succinic anhydride,ethanethiol, aluminium chloride, sodium tripolyphosphate (TPP), andglacial acetic acid of analytical grade are obtained from Sigma(Oakville, ON, Canada). Anhydrous N,N-Dimethylformamide (DMF),1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (EDC),4-(Dimethylamino) pyridine (DMAP), sodium hydroxide (NaOH), methanol,diethyl ether, chloroform, concentrated hydrochloric acid were obtainedfrom Thermo Fisher Scientific (Ottawa, ON, Canada). Thionyl chloride(SOCl₂) is obtained from VWR (Mississauga, ON, Canada). TAT peptide(NH₂-RKKRRQRRR-NH₂) (SEQ ID NO: 4) M.W. 1339.63 and MGF peptide(NH₂-YQPPSTNKNTKSQRRKGSTFEEHK-NH₂) (SEQ ID NO: 1) M.W. 2848.14, weresynthesized by Sheldon Biotech, McGill University. siRNA targeting PSN1(Accession #: NM_000021.3) targeting sequence: 5′ AAG GUC CAC UUC GUAUGC UGG (SEQ ID NO: 5) was synthesized from Dharmacon (Lafayette, Colo.,USA).

Synthesis of peptide tagged PEGylated chitosan polymer and nanoparticlespreparation. The polymer CS-PEG-TAT/MGF was synthesized as described inExample I. The nanoparticles were prepared from the chemically modifiedpolymer (CS-PEG-TAT/MGF). The formation of nanoparticles involvedionic-gelation method, as previously described by Calvo et al. (1997).The optimization of nanoparticles on the basis of size and surfacecharge has been previously performed and is published elsewhere(Malhotra et al., 1999). In brief, 0.5 mg/ml of chemically modifiedpolymer was crosslinked with 0.7 mg/ml of sodium tripolyphosphate (TPP),complexing 2 μg/ml of siRNA.

Animal model. Four weeks old Alzheimer's transgenic mice modelTg(APPSwFILon, PSEN1*M146L*L286V)6799Vas were purchased from MMRCfacility in Jackson Laboratory (Bar Harbor, Me., USA). These transgenicmice over-express both mutant human APP(695) with the Swedish (K670N,M671L), Florida (I716V), and London (V717I) Familial Alzheimer's Disease(FAD) mutations and human PS1 harboring two FAD mutations, M146L andL286V. This transgenic mice model was chosen as it over expressesamyloid-beta 42 protein in cerebral cortex and hippocampus, resulting inamyloid plaque pathology as early as 2 months of age. Othercharacteristic features include; reduced synaptic markers, increased p25levels and neuron loss.

The animals were housed in an environment with controlled temperature(22° C.), humidity, and a 12 h light/dark cycle at McGill's Animal carefacility. The animal experiment was conducted as per the protocolapproved by the Animal care committee at McGill University (Montreal,QC, Canada). Standard mouse chow pellets and water were supplied adlibitum. Animals were acclimatized for two weeks before the start of theexperiment. The animals were divided in two groups. The first group(n=4) received siRNA against PSN1 gene and the control group (n=4)received no treatment at all. The treatment began at 6 weeks of animalage. The siRNA dose administered through nanoparticles was 0.5 mg/kg ofanimal weight. Prior to the dose administration the animals wereanesthetized with isoflurane, gas anesthesia, as per the animal useprotocol of McGill University. The treatment dose was administereddaily, intranasally, for a total of 3 weeks. All the animals weresacrificed at the end of 3 weeks. Brain was harvested for PSN1 mRNAquantification.

Percentage mRNA knockdown by siRNA delivered through the nanoparticles.To further validate the knockdown results of the endogenous PS1expression in the treatment group, when compared to the control, aquantitative real time PCR was performed. Briefly, the brain tissueexcised at the end point was preserved in RNAlater® or RNALATER® RNAstabilization reagent from Qiagen and stored at −20° C., until use. Thetotal RNA was extracted using RNAeasy® or RNAEASY® Lipid tissue mini kitfrom Qiagen and the total RNA will be quantified using Nanodrop 2000spectrophotometer. The reverse transcription on total RNA was performedto obtain cDNA using a QuantiTect™ Reverse Transcription kit orQUANTITECT™ REVERSE TRANSCRIPTION KIT™ from Qiagen. A quantitative realtime PCR was performed using MBI Evolution Evagreen™ Master Mix orEVOLUTION EVAGREEN MASTER MIX™ following the manufacturer's protocol(MBI, Montreal, Canada) on ECO RT PCR machine from IIlumina. Therelative expression levels of the targeted gene were compared with thehousekeeping gene GAPDH. The primer sequences used were as follows:

PSN1: F (SEQ ID NO: 6) 5′-CCGAAATCACAGCCAAGA-3′; R: (SEQ ID NO: 7)5′-CATTCACAGAAGATACCAAGAC-3′. GAPDH, F: (SEQ ID NO: 8)5′-TAAAGGGCATCCTGGGCTACACT-3′; R: 5′- (SEQ ID NO: 9)TTACTCCTTGGAGGCCATGTAGG-3′.The PCR was run for 40 cycles with a 95° C. denaturing step (15 s), a56° C. annealing step (1 min), and a 72° C. extension step (15 s), plusfinal incubation at 72° C. for 10 min.

Evaluation of percentage gene knockdown of PSN1 gene, silenced by siRNAdelivered intranasally via novel receptor-targeted nanoparticles. ThePSN1 gene knockdown was evaluated by extracting total RNA form the braintissues, then reverse transcribing it to form a cDNA and then running aReal-Time PCR on cDNA using primers specific for PSN1 gene. Thepercentage gene knockdown of PSN1 gene was evaluated by comparing thetreatment group with the untreated control. As represented in FIG. 16 ,a 21.34% of gene knockdown was achieved in the treatment group (n=4)that received siRNA against PSN1 gene, complexed with nanoparticles, ascompared to the untreated control (n=4). Though not statisticallysignificant (p=0.162), these results propose a promising approach ofdelivering siRNA using receptor-targeted nanoparticles via intra-nasalroute for brain targeting.

Systemic safety and toxicity of novel siRNA-nanoparticle formulation.Safety markers were analyzed from the serum collected from the animalsthat received 0.5 mg/kg of PSN1-siRNA, complexed with peptide taggedPEGylated chitosan nanoparticles. The liver function and toxicity testswere performed using alkaline phosphatase (ALP) and aspartateaminotransferase/alkaline aminotransferase (AST/ALT) respectively. Theresults for ALP though show a significant difference between thetreatment and control group (FIG. 17 , p=0.036) but had values that fallin the normal range of 44 to 147 IU/L. For AST/ALT, the results indicateno significant difference between the treatment and the control group(FIG. 17 , p=0.379). Another test performed was CRP, which is anindicative of systemic inflammation the results showed no significantdifference between the treatment and the control (FIG. 17 , p=0.472).Urea, CRE and UA tests were performed indicating the renal function inanimals. The results indicate no significant difference in urea (FIG. 17, p=0.535) and UA (FIG. 17 , p=0.737) levels, however the creatinine wasfound to be elevated in the treatment group as compared to the control(FIG. 17 , p=0.025), but the values in both the groups were all found tobe in the normal range i.e. 38.13 to 91.5 μmol/L.

Example V—In Vivo Use for Treating Cancer

Cancer is characterized by uncontrolled growth of group of cells thatinfest adjacent tissues and often metastasize to other organs vialymphatic system or blood stream. Cancer is primarily caused byenvironmental factors (90-95%) and few with genetic (5-10%). Theuncontrolled growth of group of cells in the case of cancer is usuallytriggered by malfunctioning of the genes that manipulate cell's growthand differentiation. Typically the alteration in cell growth promoting,oncogenes and cell division inhibiting, tumor suppressive genes lead tothe formation of cancer cells. The genetic causes of cancer are usuallydue to gain or loss of an entire chromosome due to errors in mitosis orchanges in nucleotide leading to mutations in the genomic DNA. Dependingon the stage of the cancer the treatment options available includesurgical removal, chemotherapy with anticancer drugs, such as5-fluorouracil, oxaliplatin and leucovorin, radiation therapy,immunotherapy and hormone block therapy with drugs like cetuximab andpanitumumab. However, it has been shown that cancers with genetic originare not benefited with these chemotherapies. Moreover, the toxicity andside effects has severely limited the safety and effectiveness of thesemethods. One of the target proteins in cancer therapy is PLK1, which isa serine/threonine kinase, a key regulator for mitosis in mammaliancells. It is required for centrosome maturation, bipolar spindleformation, and chromosomal segregation and is also associated withmicrotubules and centrosomes at various stages of mitosis. PLK1 isconsidered as a proto-oncogene, which over-expresses in a variety ofhuman cancers. PLK1 is directly associated with p53, a tumor suppressorprotein. PLK1 on interaction with p53 inhibits its transactivationactivity and pro-apoptotic activity, leading to uncontrolledproliferation of cells. Lately, inhibition of PLK1 with antibodies,antisense oligonucleotides (ASO's), small interfering RNA (siRNA) ordominant negative mutants that suppress the tumor growth by causingincreased apoptosis has gained much interest as a therapeutic option totreat tumor diseases. Although, antineoplastic drugs have shown greatsuccess as a treatment for cancer therapy, many carcinomas are resistantto these agents and thus, chemotherapy with these agents has become amajor restriction at an advanced cancer stage. Therefore, siRNAstargeted against proliferation-associated signal transduction pathways,which can halt the tumor progression in animal models is emerging as anappealing approach. For cancer therapy, a receptor-targetednanoparticles for in vivo delivery of siRNA has been developed. Thetarget specificity of the nanoparticles is attributed to a peptide thatguides the nanoparticle system carrying siRNA to specific tissue, whenadministered via systemic route. The advantage of using peptide basedtumor targeting is their rapid clearance from the blood because of theirsmall size and lack of immunogenicity. The identification of peptidesfor tumor-specific targeting has been facilitated by a technologycalled, phage display library, which takes into account the ability offilamentous bacteriophage to present large number of peptides andproteins on their surface, allowing these specific peptide sequences tobind to the target specific tumors or cell types. This method foridentification of specific binding ligands has found wide application inisolating peptides that has high binding affinity towards cancer cells.The conjugation of these peptides to conventional chemotherapeuticdrugs, diagnostic/imaging molecules and nanoparticles will enable theirdelivery in low dose with effective targeting. In this study, thepeptide CP15 with a sequence NH₂-VHLGYAT-NH₂ (SEQ ID NO: 2), which wasidentified by the technology of phage displayed library was used. CP15peptide has shown to be the most effective peptide targeting colon tumorcells but not the normal human intestinal epithelial human cells. Thenovel receptor-targeted nanoparticle formulation developed in this studywere tagged with CP15 peptide as a ligand that will guide thenanoparticles to selectively target the tumor tissue expressingreceptors for CP15 peptide in a mouse xenograft model of colon cancerdeveloped from SW480 epithelial colon cancer cells. This illustrates thepotential of using these novel receptor-targeted nanoparticles to beused in future for cancer therapy.

Materials. Chitosan (M.W. 50 KDa to 190 KDa) with viscosity 20 to 300 cP(at room temperature) and degree of deacetylation of 75 to 85% wasobtained from Sigma (Oakville, ON, Canada). Polyethylene glycolmonomethyl ether (mPEG): medium molecular weight (2 000 Da), phthalicanhydride, pyridine, toluene, hydrazine monohydrate, succinic anhydride,ethanethiol, aluminium chloride, sodium tripolyphosphate (TPP), agarose,ethidium bromide (10 mg/ml) and glacial acetic acid of analytical gradeare obtained from Sigma (Oakville, ON, Canada). AnhydrousN,N-Dimethylformamide (DMF),1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) (EDC),4-(Dimethylamino) pyridine (DMAP), sodium hydroxide (NaOH), methanol,diethyl ether, chloroform, concentrated hydrochloric acid were obtainedfrom Thermo Fisher Scientific (Ottawa, ON, Canada). Thionyl chloride(SOCl₂) is obtained from VWR (Mississauga, ON, Canada). CP15(NH₂-VHLGYAT-NH₂) (SEQ ID NO: 2) M.W. 758.3, were synthesized by SheldonBiotech, McGill University. Pre-cast NUPAGE™ 4 to 12% Bis-Tris gels,Nupage MES running buffer, NUPAGE™ transfer buffer, NUPAGE™ LDS samplebuffer (4×), nitrocellulose membrane 0.45 μm, blotting filter paper andMagic Mark™ or MAGIC MARK™ (1 Kb) protein ladder were obtained fromInvitrogen (Burlington, ON, Canada). Scrambled/non-targeting (NT) siRNAtagged with biotin, and PLK siRNA (h) with sequence; PLK (sensestrand)-5′ AGAmUCACCCmUCCUmUAAAmUAUU 3′ (SEQ ID NO: 10) and PLK(antisense strand)-5′ UAUUUAAmGGAGGGUGAmUCUUU 3′ (SEQ ID NO: 11) fromDharmacon (Lafayette, Colo., USA), where “m” represents 2′Omethylatednucleotide. Primary antibodies; PLK1 (F-8) mouse monoclonal antibody,β-Actin (C-4) mouse monoclonal antibody, probed with secondaryantibodies; (HRP)-conjugated goat antimouse antibody. All the antibodieswere procured from Santacruz Biotechnology (Santa Cruz, Calif., USA).

Synthesis of peptide (CP-15) tagged PEGylated chitosan polymer. Thepolymer CS-PEG-CP15 was synthesized as described in Example I. Themodified polymer was characterized and analyzed using ¹H (Mercury 400and 500 MHz NMR) and Transmission Electron Microscopy (Philips EM410TEM).

Preparation of novel polymeric nanoparticles from CP-15 tagged PEGylatedchitosan polymer. Nanoparticles were prepared from the chemicallymodified polymer (CS-PEG-CP-15). The formation of nanoparticles involvedionic-gelation method, as previously described by Calvo et al. (1997).The optimization of nanoparticles on the basis of size and surfacecharge has been previously performed and is published elsewhere(Malhotra et al., 2009). In brief, 0.5 mg/ml of chemically modifiedpolymer was crosslinked with 0.7 mg/ml of sodium tripolyphosphate (TPP),complexing 8 μg/ml of siRNA. The characterization of nanoparticles sizeand dispersivity was analyzed by transmission electron microscopy (TEM)and the siRNA loading efficiency was determined by gel retardationassay.

Animal study and in vivo tumor induction. Six weeks old mice Balb/c nudemice, weighing 15 to 20 g were purchased from Charles River Laboratories(Wilmington, Mass., USA) and housed in an environment with controlledtemperature (22° C.), humidity, and a 12 h light/dark cycle at McGill'sAnimal care facility. The animal experiment was conducted as per theprotocol approved by the Animal care committee at McGill University(Montreal, QC, Canada). Standard mouse chow pellets and water weresupplied ad libitum. Animals were acclimatized for a week before thestart of the experiment. For tumor induction, animals weresubcutaneously inoculated with 100 μl of SW480 colon cancer cells(2×10⁶) mixed with an equal volume of MATRIGEL™ (BD). The treatmentbegan after the tumor reached a volume of 100 mm³. The animals wererandomized into 4 treatment groups (n=7) to receive treatmentformulations. The following treatment formulations: 1) CS-PEG-CP15 withPLK1 siRNA; 2) CS-PEG-CP15 with non-targeting (NT) siRNA, 3) PLK1 siRNAalone and 4) Untreated control. In each treatment group the animalsreceived a total siRNA dose of 0.5 mg/kg. 100 μl of treatmentformulations were administered thrice a week via intra-peritonealinjections for a period of 2 weeks.

Histopathological analysis to identify siRNA delivered via novelnanoparticles in tumor tissue. One animal from each treatment group waseuthanized by CO₂ asphyxiation, after 4 hours of first dose administeredvia intra-peritoneal route. Brain, Lungs, Heart, Kidney, Spleen andLiver were harvested and kept at 4° C. in 10% phosphate bufferedformalin for 48 h. After which the tissues were trimmed to 3 mm thicksections and stored at 70% ethanol in histology cassettes. The tissueswere paraffin embedded and processed into 4 μm thick section on slidesat the histology core facility (The Rosalind and Morris Goodman CancerResearch Centre, McGill University). The tissue section on slides werestained with Vectastain Elite ABC™ kit (Vector laboratories; Burlingame,Calif., USA) as per the manufacturer's protocol and Diaminobenzidine(DAB) was used as a substrate to assess the presence of biotin, whichwas used as a tag on siRNA for histology identification purposes.Hematoxylin was used as a counterstain and slides were mounted withPERMOUNT™ (Vector laboratories; Burlingame, Calif., USA) and observedunder compound microscope (Leica DM500; Ontario, Canada) at ×400magnification.

RNA extraction and QPCR to determine percentage gene knockdown by siRNAtargeted against PLK1 gene delivered through novel nanoparticles. Tofurther validate the knockdown effects of endogenous PLK1 expressionafter nanoparticles based siRNA delivery against PLK, a quantitativereal time PCR was performed. Briefly, the tumor tissues excised at theend point were preserved in RNAlater® RNA stabilization reagent fromQiagen (Toronto, ON, Canada) and stored at −20° C. The total RNA wasextracted using RNeasy® Plus mini kit from Qiagen, following themanufacturer's protocol. The total RNA was quantified using Nanodrop2000 spectrophotometer from Thermo Scientific (Rockford, Ill., USA).Reverse transcription was performed to obtain cDNA from 1 μg of totalRNA using a QuantiTect™ Reverse Transcription kit or QUANTITECT™ REVERSETRANSCRIPTION KIT™ from Qiagen. Following that quantitative real timePCR was performed using MBI Evolution Evagreen Master Mix™ or EVOLUTIONEVAGREEN MASTER MIX™ following the manufacturer's protocol (MBI,Montreal, Canada) on ECO RT™ PCR machine from IIlumina (San Diego,Calif., USA). The relative expression levels of PLK1 gene werenormalized with the housekeeping gene GAPDH. The primer sequences usedwere as follows:

PLK1 Plk1, (SEQ ID NO: 12) 5′-GGCAACCTTTTCCTGAATGA-3′ and(SEQ ID NO: 13) 5′-AATGGACCACACATCCACCT-3′; GAPDH, (SEQ ID NO: 14)5′-TAAAGGGCATCCTGGGCTACACT-3′ and (SEQ ID NO: 15)5′-TTACTCCTTGGAGGCCATGTAGG-3′.The PCR was run for 30 to 40 cycles with a 95° C. denaturing step (5 s),a 60° C. annealing step (15 s), and a 72° C. extension step (15 s), plusfinal incubation at 72° C. for 10 min.

Protein expression after siRNA delivered through novel nanoparticles.After 2 weeks of treatment, the animals were sacrificed as per theapproved protocol by McGill University and the tumor tissues wereharvested and preserved at −20° C. in ALL PROTECT TISSUE REAGENT™ fromQiagen (Toronto, ON, Canada). The tissue samples were sliced into smallpieces and homogenized using POWERGEN MODEL 125™ Homogenizer from FisherScientific (Ottawa, Ontario, Canada) at 26 300 rpm in 2 mL of ice coldRIPA buffer (20 mM Tris pH 8, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40,0.1% SDS, 10.0% Glycerol, 10 mM Na₂HPO₄·7H₂O, 1% Sodium deoxycholate)containing phenylmethylsulfonyl fluoride (PMSF) and protease inhibitorcocktail from Roche Diagnostics (Laval, QC, Canada). The crude extractwas incubated on ice for 30 minutes and centrifuged at 10 000×g for 10minutes at 4° C. to remove tissue debris. The supernatant was collectedand the protein concentration was determined using Pierce® BCA Proteinassay kit from Thermo Scientific (Rockford, Ill., USA). Briefly,aliquots containing 100 μg of protein were heated at 70° C. for 15minutes with Nupage LDS sample buffer supplemented with 100 mM DTT. Theproteins were fractionated on precast NuPAGE® 4 to 12% Bis-Tris Gel fromInvitrogen (Ontario, Canada) at 200 V for 35 minutes in MES SDS runningbuffer. Magic Mark™ 1 Kb protein ladder was used as a standard. The gelwas electrophoretically transferred to 0.45 μm pore size Novex®nitrocellulose membrane using Nupage transfer buffer on a Novex® SemiDryblotter (Invitrogen, ON, Canada). After transfer the nitrocellulosemembrane was incubated for 1 hr in 5% non-fat powdered milk in Trisbuffered saline (TBS) buffer supplemented with 0.2% Tween 20. Themembrane was then incubated overnight at 4° C. with mouse PLK (F-8)monoclonal antibody (1:100 dilution). The next day the membrane waswashed thrice with TBST for 15 minutes each, and then incubated with(HRP)-conjugated goat antimouse IgG secondary antibody (1:2,000dilution). The membrane was again washed thrice with TBST followed bydetection of signal with chemiluminescent agents (ECL, Amersham) from GEhealthcare. The bound antibody was visualized using autoluminography. Tocontrol the protein loading, the membrane was reprobed with primarymouse monoclonal β-Actin (C-4) antibody (1:1 000) with an overnightincubation at 4° C., followed by subsequent three washes in TBST anddetection with HRP-conjugated goat anti-mouse IgG secondary antibody(1:2 000) and development with chemiluminescent agents as describedearlier. The protein band intensities were quantified using IMAGE J™software.

Serum collection and analysis. Serum was collected through jugular veinjust before the end-point using a sterile 23 G/25 mm needle andapproximately 200 μl of blood was collected in Microtainer® serumseparator tubes (Becton Dickinson, N.J., USA). The blood was allowed toclot at room temperature for 30 minutes and subsequently placed on iceuntil centrifugation. Serum was separated by low-speed centrifugation at3600 rpm for 8 min at 4° C. The separated serum was frozen at −85° C.until analysis. Serum was used to test C-reactive protein (CRP) andliver function tests such as alanine aminotransferase (ALT) andaspartate transaminase (AST) using a conventional enzymatic method onHitachi 911 automated clinical chemistry auto-analyzer (RocheDiagnostics, USA).

Statistical analysis. Statistical analysis was carried out usingGRAPHPAD PRISM™ version 5.0 for windows (GraphPad software, San Diego,Calif.). Values are expressed as means±Standard Error. The statisticalcomparison between treatment groups was performed using ANOVA Tukey'stest. P values<0.05 were considered significant.

Synthesis of CP-15 tagged PEGylated chitosan polymer and preparation ofnanoparticles. The peptide-tagged PEGylated chitosan polymer wassynthesized following a series of chemical reactions as represented inFIG. 1 . Each intermediate step and the final product of the synthesiswere characterized for the functional group modification andsubstitution by FTIR and ¹H NMR as optimized in Malhotra et al. (2009).FIG. 18 illustrates the NMR spectra of the final product obtained(CS-PEG-CP15) after the synthesis. The multiple peaks of oxymethylgroups in PEG at δ 3.3 to 3.7 cover over the signals of pyranose ring ofchitosan in the spectra. The weak and broad peak at δ 4.3-4.5 are fromthe protons of —NH—CH(CH₂)—CO— in CP-15 peptide. The multiple peaks at δ6.0-9.0 belong to the CP-15 peptide sequence.

Characterization of nanoparticles. Nanoparticles were prepared followingan ionic gelation scheme, wherein the cationic polymer complexes theanionic molecule due to electrostatic interaction. FIG. 19 representnanoparticles as observed under TEM. The nanoparticles obtained werepolydispersed and ranged from 100 to 200 nm in size. Thepolydispersivity observed in nanoparticles size depends on the averagemolecular weight of the polymer used. Molecular weight corresponds tothe chain length of the polymer. In this example, chitosan of molecularweight 50 KDa to 190 KDa was used as a parent polymer on which furtherchemical modifications and substitution of a hydrophilic polymer, PEGand a cell targeting/penetrating peptide (CP15) was performed. Since,each monomer of a chitosan polymer comprises of an amine group, thuslarger chain length contributes to more number of amine groups. Thepresence of amine groups adds a positive character to the polymer, whichenables it to complex a negatively charged molecule, such as siRNA. Dueto the increase in the polymer chain length of chitosan as compared tothe other low molecular weight chitosan (M.W. 10 KDa) used for brainstudy (Example I to III), it was anticipated that the nanoparticleformulation will be able to complex more siRNA. To confirm thisassumption, a gel retardation assay was performed (FIG. 20 ) and it wasobserved that the nanoparticle formulation was able to complex 4 timeshigher amount of siRNA (8 μg/ml) than the previous formulation (2μg/ml).

Evaluation of the ability of the receptor-targeted nanoparticles tosystemically deliver siRNA at the targeted tumor site. Novelreceptor-targeted nanoparticles were evaluated for their ability todeliver siRNA at the targeted tumor site through systemic route. Theanimals were sacrificed after 4 hours of first dose administeredintra-peritoneally. The treatment formulation prepared from a chemicallymodified polymer, CS-PEG-CP15 was complexed with scrambled biotinylatedsiRNA at 0.5 mg/kg and was evaluated for its efficiency in comparison tosiRNA delivered without nanoparticles. FIG. 21 representshistopathological staining from a mouse xenograft model of SW480 coloncancer. The tumor tissue having the scrambled biotinylated siRNA staineddark brown in color with VECTA™ stain elite kit, using DAB as asubstrate. The results shown on FIG. 21(I) indicate that the stainingwas found to be approximately equal for both (a) CS-PEG-CP15-siRNAnanoparticle formulation (p=0.00043) and (b) unmodified chitosan-siRNAnanoparticle formulation (p=0.0011). The expression of (c) scrambledbiotin-siRNA, delivered alone was comparatively less and was notsignificant, when compared to the untreated control (p=0.062) FIG. 21(II) represents the mean percentage area analysed for intensity intriplicates from an animal tissue in each treatment, using IMAGE J™software.

FIG. 22A represents the biodistribution analysis of the above mentioned3 treatments in various other organs: heart, lungs, kidney, liver andspleen and were compared with the untreated control. The resultsindicate significant biodistribution of the unmodified chitosannanoparticles in all the organs (p<0.05) when compared with the control.For CS-PEG-CP15 with siRNA nanoparticle formulation, the biodistributionwas found to be significant only in heart (p=0.001) and lungs (p=0.017),as compared with the control. Likewise, siRNA delivered alone alsoshowed significant expression in heart (p=0.009) and lungs (p=0.043).FIG. 22 (II) represents the mean percentage area analysed for intensityin duplicates from an animal tissue in each treatment, using IMAGE J™software.

The tumor accumulation data as represented in FIG. 21 , suggests thatthe derivatized chitosan nanoparticles were able to target the tumortissue as effectively as the unmodified chitosan nanoparticles, whencompared with the siRNA delivered alone and the untreated control. Thisdata suggests that the importance and efficiency of chitosan polymer asa carrier to deliver the siRNA at the targeted site. Thus, it can beinferred that the systemic tumor targeting, could majorly be a sizedependent phenomenon due to the EPR effect rather than receptor mediatedtargeting. However, the biodistribution study as presented in FIG. 22showed that the CS-PEG-CP15 nanoparticles accumulated less in otherorgans, when compared to the unmodified chitosan nanoparticles (kidneys,p<0.046). Thus, it can be inferred that the presence of a targetingmoiety on the nanoparticles restricted their uptake by other tissues[41]. The enhanced circulation and lack of accumulation in other organsof CS-PEG-CP15 nanoparticles is attributed to the incorporation of PEG,which caters to the increased stability of the nanoparticles in blood,without being degraded or filtered by kidneys. These results indicatethat these nanoparticles have the ability to be administered vianon-invasive routes and at the same time achieve effective tumortargeting, which in turn will reduce the off-target effects and unwantedimmunological reactions and toxicity.

Evaluation of percentage gene knockdown of PLK1 gene, silenced by siRNAdelivered systemically via novel receptor-targeted nanoparticles. ThePLK1 gene knockdown was evaluated by extracting total RNA form the tumortissues, then reverse transcribing it to form a cDNA and then running aReal timer PCR on cDNA using primers specific for PLK 1 gene. Thepercentage gene knockdown of PLK1 gene by siRNA delivered through novelreceptor-targeted nanoparticles was evaluated by comparing the treatmentgroup with other controls; mock transfections and untreated. Asrepresented in FIG. 23 , it was observed that the treatment group showeda significant reduction (P=0.031) in PLK1 gene knockdown of 50% ascompared with the untreated control (n=6). There was no significantdifference in the group of animals that received mock transfections i.e.nanoparticles with NT siRNA (P=0.933) and PLK1 siRNA alone (P=0.539),when compared with the untreated control. This result confirmed that thenanoparticles were able to safeguard the siRNA when administered viasystemic route and delivered it specifically at the tumor site. Underthese experimental conditions, the siRNA retained its functional abilityof gene knockdown and was not degraded by the serum proteins.

Evaluation of protein suppression of PLK1 protein by siRNA, deliveredsystemically via novel receptor-targeted nanoparticles. To evaluate andcompare the amount of protein expression in various treatment groups,the total protein was extracted from the tumor tissue, quantified usingBCA assay and 100 μg of total protein was loaded onto NuPAGE® 4 to 12%Bis Tris gels for western blot analysis. The gel was electrophoreticallytransferred to a nitrocellulose membrane and probed with appropriateantibodies. The protein bands developed using autoluminiscence werequantified using an Image J software and plotted with animal numbers,n=6 in each group. The relative protein expression as observed in FIG.24 shows that the animals receiving novel receptor-targeted nanoparticleformulation carrying siRNA against PLK1 gene has significantly lowerprotein expression (P=0.038) as compared to the control, untreatedgroup. However, no difference was observed among the mock transfectiongroups i.e. nanoparticles with NT siRNA (P=0.999) and PLK1 siRNA alone(P=0.758) when compared with the control untreated group. These resultssuggest that the siRNA targeted against PLK1 was solely responsible forthe decrease in the protein expression in the tumor tissue and there wasno deleterious effect posed by the polymeric nanoparticle formulation onthe tumor tissue.

Serum analysis for safety and toxicity study. 200 μl of blood wascollected in microtainer serum separating tubes just before theexperimental end point, from jugular vein of all the animals in eachgroup. The experimental end-point was at 2 weeks after the commencementof the treatment formulation. The serum was analyzed for safety testsespecially for liver function tests (AST/ALT) and C-reactive proteins(CRP) as represented in FIGS. 25A and 25B respectively. No significantdifference was observed among any treatment groups when compared to theuntreated control for both CRP and AST/ALT ratios. Thus, these resultsindicate that the receptor-targeted nanoparticle formulation was nottoxic and does not stimulate immunological reactions. It also shows thatthe percentage gene knockdown and protein suppression as observed inFIGS. 23 and 24 is the result of gene knockdown mediated by the siRNAtargeted against PLK1 gene leading to cell apoptosis and was not due toany deleterious/toxic effects of the polymeric nanoparticle formulationused as a transfection reagent.

The results presented herein show the potential of novelreceptor-targeted nanoparticle formulation to be used in vivo in a mousexenograft model of colon cancer. These nanoparticles prepared from achemically modified polymer have the ability to be modified byincorporation of a specific peptide or a cell targeting ligand/moiety,which can target a specific receptor on a cell. The nanoparticlesprepared showed efficient siRNA complexation and delivery withapproximately 100% transfection efficiency. The nanoparticles safeguarded the siRNA when administered systemically and specificallyreleased it at the tumor site. 50% of gene knockdown of PLK1 wasachieved as determined by qPCR analysis with siRNA delivered throughnovel receptor-targeted nanoparticles. These nanoparticles did notinduce any immunological reactions and liver toxicity as determined bythe serum analysis. This study proves that nanoparticles mediated genetherapy can be achieved and performed via non-invasive strategies. Thesenanoparticles as a delivery device can further be explored towardsdifferent applications such as cancer and neurological disorders. Thisstudy can further be improved by prolonging the treatment duration andincreasing animal number per group in order to achieve a significantdifference and refined interpretation of the data.

Example VI—Subject Study for Treating Cancer

Overexpression of Plk1 has been observed in a number of human cancersincluding non-small-cell lung cancer, head and neck cancer, esophagealcancer, gastric cancer, melanomas, breast cancer, ovarian cancer,endometrial cancer, colorectal cancer, glioma, papillary carcinoma,pancreatic cancer, prostate cancer, hepatoma, leukemia and lymphoma,bladder cancer, and thyroid cancer. Many of these studies havedemonstrated that Plk1 overexpression correlates with tumor progressionand patient survival rate in a variety of cancers. Therefore, Plk1 isproposed as a prognostic marker for human cancers.

A subject study was performed on a patient with Stage IV head and neckcancer. For this study, the synthesis of polymer was scaled up to obtain2 g of synthesized polymer, which was used to form siRNA nanoparticlesfor the treatment of cancer. The siRNA used was against PLK1 mRNA, whichis a proto-oncogene. The therapy was termed “Nora-PLK1”.

The scaling up procedure to synthesize the polymer was performed underaseptic conditions following Good Laboratory Practices (GLP). Followingis the Standard Operating Procedure (SOP) as detailed herein,

-   -   1) 4 g of Chitosan LMW with 11.04 g of phthalic anhydride in 80        ml of DMF. Stirred for 8 hrs at 110° C. under N₂ atmosphere.        Precipitate it in ice cold water and give a methanol wash        overnight. Vacuum filter next morning using a whatman filter        paper on a funnel and vacuum dry. The polymer will appear light        brown in color). Product “c” in FIG. 1 . (Phthalic anhydride        should be 3 times mole excess of chitosan)    -   2) 60 g of PEG (M.W. 2000) dissolved in 200 ml toluene with 12 g        of succinic anhydride dissolved in 40 ml of pyridine. Stir and        reflux the reaction at 100° C. for 6 hrs. Precipitate with        diethyl ether and vacuum filter using a whatman filter paper on        a funnel. Dissolve the product back with chloroform and again        precipitate with diethylether, followed by vacuum filter on a        whatman filter paper using a funnel. Repeat this process twice.        At the end vacuum dry the product. (The product will appear        white in color). Product “a” in FIG. 1 . (Succinic anhydride        should be 4M excess of PEG). (Note: Repeat this step in batches        for scale up if required).    -   3) To the dried PEG from step 2, dissolve it in 200 ml of        toluene and add equimolar of thionyl chloride. Stir and reflux        the reaction at 100° C. for 6 hrs, followed by degassing to        remove excess SO₂ and thionyl chloride. Product “b” in FIG. 1 .    -   4) After 6 hrs, from step 3, bring down the reaction to room        temperature (RT) and add phthaloyl chitosan from step 1 (pre        dissolved overnight in pyridine). 1.8 g of chitosan        (pre-dissolved in 50 ml of pyridine) was reacted with 60 g of        PEG2000 (predissolved in 200 ml of toluene). (As the chitosan        (dissolved in pyridine) is added to the PEG (toluene) solution.        The reaction will look heterogeneous).    -   5) Let the reaction continue for 2 hrs at RT and then reflux at        100° C. for 24 hrs. After 24 hrs, bring the reaction down to RT        and precipitate in methanol and vacuum filter dry on a whatman        filter paper using funnel. (The product will appear pale brown        in color). Product “d” in FIG. 1 . (PEG2000 should be 10M excess        of phthaloyl chitosan).    -   6) The filtered product from step 4, mix 3 g of PEGylated        phthaloyl chitosan with 174 mg of Aluminum chloride in 80 ml of        ethanethiol. Let the reaction stir at 23-25° C. for 12 hrs.        After the reaction, dilute it with water and acidify with 10%        HCl. Vacuum Filter the product on a whatman filter paper using        funnel. Mix the resultant product with deionized water transfer        it to a separating funnel. Extract the purified product in the        separating funnel using dichloromethane three times. Rotavap the        dichloromethane and obtain the dried product. (The product will        appear brown and sticky). Product “e” in FIG. 1    -   7) To the dried product from step 5, 2.6 g of PEGylated chitosan        was dissolved in 100 ml of toluene and mixed with 452.35 mg of        succinic anhydride (predissolved in 5 ml of pyridine). The        reaction is stirred at 100° C. for 12 hrs. After the reaction        the product brought down to RT and is precipitated in methanol        and vacuum filtered (whatman paper and funnel) and vacuum dry.        (The polymer will appear dark brown solid in color). Product “f”        in FIG. 1 . (Succinic anhydride should be 4M excess of PEGylated        chitosan).    -   8) Take 1 g of the dried CS-PEG-COOH from step 6 and dissolve it        in 25 ml of DMF. Add 1.3 mole equivalent of EDC.HCl to it (87.5        mg) and 0.1 mole equivalent of DMAP (5.3 mg). Dissolve 500 mg of        TAT peptide in 5 ml of DMF and add it to the above mixture. Stir        it for 24 hrs at room temperature. Precipitate the mixture in        ice cold water and filter (whatman paper and funnel), give the        filtrate a brief wash in methanol and again filter dry it. (The        product will appear brown in color). Product “g” in FIG. 1 .    -   9) Take 1 g of the dried product from step 7 and dissolve it in        20 ml of DMF and dissolve at 80° C. under inert conditions,        after that add 2.6 ml of hydrazine monohydrate to it and let the        reaction stir at similar conditions for 2 hrs. (The product will        appear pale yellow-white in color). Product “h” in FIG. 1 .        Bring the reaction to room temperature and Dialyze the product        using slide-a-lyzer (dialysis tube) MW cut off of 3,500 for 2-3        days in sterilized water. While doing dialysis change the water        at least 3-4 times a day. After dialysis, concentrate the        product to remove excess water by vacuum filter (whatman paper        and funnel) and/or freeze dry the sample, overnight and you will        obtain an off-white colored fluffy polymer.    -   10) To prepare Nora-PLK1 nanoparticles, dissolve the synthesized        Lyophilized polymer at a concentration of (0.5 mg/ml) in a        sterilized water, containing 2% acetic acid and adjust the pH to        5 using 5M NaOH solution. Stir this solution overnight at 50° C.        Filter the polymer solution using sterile STERICUPS™ (filters)        with pore size of 0.2 μm.    -   11) Mix 2 μg of siRNA (20 μM siRNA stock) to the polymer (0.5        mg/ml) solution. [To prepare a dose of 50 μg of siRNA. Aliquout        25 ml of filtered chitosan polymer solution in a falcon tube and        add 195 μl of (20 μM siRNA stock) and vortex vigorously for 5-10        seconds and keep at bench for 30 minutes for complexation and        stabilization.    -   12) The nanoparticles after complexation with siRNA, be filtered        through a sterile 0.45 μm filters. The sterile filtered        nanoparticles should be concentrated using Am icon centrifugal        concentrators (MW. Cut-off of 30,000) and spun down at 4000 rpm        for 30-40 minutes to obtain a concentrate of 1.5 to 2 ml volume.        (While concentrating care must be taken that the nanoparticles        do not precipitate or aggregate)..    -   13) For freeze-drying, the concentrated siRNA-nanoparticles        should be transferred to autoclaved eppendorf tubes and 300 μl        of 60% sucrose solution should be added. The concentrated        siRNA-nanoparticle solution, containing sucrose should be stored        in −20° C. for 30 minutes, followed by freezing in −80° C. for        1-2 hrs and be placed in a lyophilizer for overnight drying.        After lyophilisation, the siRNA-nanoparticle material can be        stored at −80° C. until use or can be reconstituted in        sterilized water up to 0.5 to 1 ml for delivery (intra tumor or        intra venous) purposes.    -   14) The synthesis proved to be reproducible after being scaled        up. The average nanoparticle size was <200 nm with a zeta        potential of 16-20 mV.

The subject study was performed on female, aged 55 years old. Thepatient had Stage IV head and neck cancer, specifically called squamouscell carcinoma of tonsil. The patient had sub-mandibular lymph nodemetastasis and smaller in the right neck. There are 3 small metastasisin as many left ribs and multiple lung metastasis in both lungs. Thepatient had no prior treatment with chemo or radio therapy. The majoroutcome of the study was based on the reduction in the size of thetumors based on the CT scans. The intervention used for this studyincluded TAT peptide-tagged PEGylated chitosan nanoparticle carryingPLK1 siRNA, termed as “Nora-PLK1” therapy.

The intervention included a total dose of 100 μg (siRNA) of Nora-PLK1therapy, administered locally via intra-tumoral injections at alternatesites in the neck and tongue and/or via inhalation using anebulizer/inhaler to reach lungs. The Nora-PLK1 therapy was initiated onSep. 28, 2014 and was given for a total of 6 weeks, everyday excludingweekends.

The intratumoral application resulted in a dramatic reduction of thetumor mass. Volumetrically the tumor reduced from 29.66×41.66×46 mm to18.66×35.55×41 mm or from 56,839.2 m³ to 27,197.8 m³ (a 46% reduction)for the first 10 day therapy cycle of the Nora-PLK1 treatment (FIG. 26and FIG. 27 ). Followed by complete disappearance of tumor mass byDecember 2014.

Complete disappearance of the tumor mass from lungs was observed (CTscans) with Nora-PLK1 therapy administered via inhalation using anebulizer (FIG. 28 ).

The successful results obtained from the subject study showed, improvedhealth benefits such as, disappearance of cancer from neck and lungs,improved speech, ability to eat and swallow food, gain in body-weight.The therapy worked without the need of any invasive techniques such assurgery or radio/chemotherapy. The present invention represents aplatform technology, wherein the peptide used in the polymeric synthesiscan be replaced with any other cell-targeting peptide, protein,antibiotic, antibody, pharmaceutical compounds. The therapeutic payload,complexed with the nanoparticle is not limited to siRNA, but caninclude, pDNA, miRNA, small oligonucleotides, chemotherapeutic drugs,nutraceutical compounds. The nanoparticles produced can also beformulated in a hydrogel for topical application purposes, or mixed withmicroparticles for oral delivery purposes to provide a varied range ofits health beneficial effects.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features herein before set forth, and as follows in the scopeof the appended claims.

REFERENCES

-   Calvo, P, Remunan-Lopez, C, Vila-Jato, J L, Alonso, M J: Novel    hydrophilic chitosan-polyethylene oxide nanoparticles as protein    carriers. J Appl Polym Sci 63:125-132, 1997.-   Kurita K, Ikeda H, Yoshida Y, Shimojoh M, Harata M: Chemoselective    protection of the amino groups of chitosan by controlled    phthaloylation: facile preparation of a precursor useful for    chemical modifications. Biomacromolecules 3:1-4, 2002.-   Lin W J, Chen M H: Synthesis of multifunctional chitosan with    galactose as a targeting ligand for glycoprotein receptor.    Carbohydrate polymers 67:474-480, 2007.-   Malhotra, M, Kulamarva, A, Sebak, S, Paul, A, Bhathena, J, Mirzaei,    M, Prakash, S: Ultrafine chitosan nanoparticles as an efficient    nucleic acid delivery system targeting neuronal cells. Drug    Development and Industrial Pharmacy 35:719-726, 2009.

What is claimed is:
 1. A method for in vivo targeted delivery of anucleic acid or an anionic agent to an individual in need thereof, saidmethod comprising: (a) providing a polymeric delivery system comprisingthe nucleic acid or the anionic agent, wherein the polymeric deliverysystem comprises non-viral nanoparticles comprising: a plurality ofcationic chitosan polymers, polyethylene glycol and a peptide sequence,and wherein the non-viral nanoparticles comprise an internal core andthe nucleic acid or the anionic agent are placed in the inner core, andwherein the peptide sequence comprises a cell penetrating peptide, aprotein transduction domain, and a cell surface receptor targetingmoiety, and the polymeric delivery system is made by a methodcomprising: a) reacting amine groups of chitosan with phthalic anhydrideas a protection step; b) activating monomethoxy polyethylene glycol(mPEG-OH) with succinic anhydride to form mPEG-COOH; c) activatingmPEG-COOH with thionyl chloride to form mPEG-COCl to form an activatedPEG; d) reacting hydroxyl groups of chitosan with the activatedmPEG-COCl to form PEGylated chitosan; e) reacting the monomethoxy groupof the PEGylated chitosan with aluminum chloride in ethane thiol toconvert the monomethoxy group to a hydroxyl group to form hydroxylterminated PEGylated chitosan; f) reacting the hydroxyl terminatedPEGylated chitosan with succinic anhydride to form an activatedchitosan-PEG-COOH; g) reacting the activated chitosan-PEG-COOH with apeptide sequence comprising a cell penetrating peptide, a proteintransduction domain, and a cell surface receptor targeting moiety,wherein said peptide sequence comprises a terminal amine, and whereinthis reaction is done using EDC and DMAP as a catalyst to formchitosan-PEG-peptide; and h) reacting the chitosan-PEG-peptide withhydrazine monohydrate to deprotect amine groups of chitosan, and whereinthe plurality of cationic chitosan polymers are cross linked to eachother to form the outer layer of the nanoparticle to generate aninternal core; and the nanoparticles have an average diameter of between5 to 300 nm; and (b) administering the non-viral nanoparticle to theindividual.
 2. The method of claim 1, wherein the nucleic acid oranionic agent is delivered to the individual in need thereof to treat adisease or a condition.
 3. The method of claim 2, wherein the disease orcondition is a cancer or a neurodegenerative disease.
 4. The method ofclaim 3, wherein the cancer is a brain cancer or colon cancer.
 5. Themethod of claim 1, wherein the nucleic acid is a siRNA.
 6. The method ofclaim 5, wherein the siRNA is a Plk1-targeting siRNA.
 7. The method ofclaim 1, wherein the nanoparticles have an average diameter of between100 nm to 200 nm.
 8. The method of claim 1, wherein the averagemolecular weight of the cationic chitosan polymers is from about 50 kDato about 200 kDa.
 9. The method of claim 1, wherein the degree ofdeacetylation of the cationic chitosan polymers is about 80%.
 10. Themethod of claim 1, wherein the nanoparticles have an average diameter ofless than 20 nm.
 11. The method of claim 1, wherein the nanoparticlesare formulated for intranasal administration and the administration isintranasal.
 12. The method of claim 1, wherein the nanoparticles areformulated for intravenous administration and the administration isintravenous.
 13. The method of claim 1, wherein the non-viralnanoparticle is formulated for intratumoral administration and theadministration is intratumoral.